Compound binding to leukocytes and medicinal composition containing the compound in labeled state as the active ingredient

ABSTRACT

A compound binding to leukocytes, which comprises Met-Leu-Phe or Nle-Leu-Phe serving as the leukocyte-binding site of a formyl peptide receptor (FPR), a binding part comprising Ser or Thr elevating the binding ratios to monocytes and lymphocytes in all leukocytes, a group which can be labeled with a radioactive metal or a paramagnetic metal, and a spacer binding them shows binding properties specific to all leukocytes, i.e., neutrophils, monocytes and lymphocytes both in vivo and in vitro and can be labeled with a radioactive metal or a paramagnetic metal. Owing to these characteristics, this compound is highly useful in SPECT image diagnosis, PET image diagnosis, MRI image diagnosis and so on wherein imaging is performed in a site with vigorous leukocyte infiltration accompanied by an immune reaction in an individual.

TECHNICAL FIELD

The present invention relates to a compound binding to leukocytes and amedicinal composition containing the compound in labeled state as theactive ingredient which can be used for targeting abnormal site as wellas for understanding precisely a state of disease activity when adisease of an individual associated with immune reaction is to bediagnosed and/or treated. More in detail, the present invention relatesto a novel compound having binding properties specific to leukocytesboth in vivo and in vitro and can be labeled with a radioactive metal ora paramagnetic metal, which is useful for pathological imaging of a seatof disease including infectious diseases, inflammation, tumor andatheroscrelosis in the body of mammals. Also, the present inventionrelates to a medicinal composition containing said compound in labeledstate as the active ingredient which is useful for radioisotopediagnosis, SPECT image diagnosis, PET image diagnosis, MRI imagediagnosis or radiotherapy.

PRIOR ART

Animals including human are always influenced by the factors that mayaffect on the life supporting systems of an individual from thesurrounding environment. These include, for example, factors withpositive effects such as air, sunlight and foods, and factors withnegative effects such as invasion of microorganisms, hazardous chemicalsubstances, heat and radiation. Against the factors with such negativeeffects, the individuals brings various protecting systems into actionto keep their lives.

This self-protecting system is defined biologically as immunity, and abiological reaction in relation to the immunity is referred to as immunereaction. As the factors bringing on such reactions, for example,microorganisms such as bacteria and mycoplasma, viruses, heterografts oftissue or organ, hazardous chemical compounds, heat of high temperature,excessive cooling, nuclear radiation with high energy, and electric orphysical injury of tissue are known. The immune reaction includinginflammation reaction is a biological reaction in response to therecognition of “self” or “not self” for the individual, which is anaction of the protection system in a broad sense such as fever,leukocytes activation and migration, elimination of all of the factorsexcept “self”. Inflammation reaction is a phenomenon appeared as a partof results of the immune reactions such as removal of extraneoussubstances infiltrated into an individual, demolition of invaded tissueand restoration of injured tissue.

Leukocytes are included as one of important factors in the immunereactions. Tissues produce various mediators which specify the speciesof infiltrating leukocytes and control its level and duration, and alsohave, on the surface of cell membrane, various receptors which mayrespond by binding to mediators and other molecules existing in the bodyfluid including blood. The receptor activates leukocytes by binding tothe corresponding mediator, and different types of receptors areexpressed specifically depending on the species of leukocyte. Therefore,the species of leukocytes which infiltrate into the tissue is governedby the existing mediator.

Generally, in a local immune response such as inflammation, aftergetting something stimulus of tissue demolition, proteins relating toimmunity such as complements take place to remove the stimulus forseveral hours. After that, mediators like the decomposed complementsand/or cytokines are released from the demolished tissue into body fluidsuch as blood, and granulocytes mainly composed of neutrophils areactivated thereby through the receptor, so that the leukocyteinfiltration into the tissue takes place according to a density gradientof the mediator. In such case, the mediator is generally referred to asa chemotactic factor. Normally, infiltration of tissue by granulocytesreaches to a peak at over ten hours after initial stimulus. Also,infiltration by macrophages mainly composed of monocytes increasesgradually to remove the cause substance of the stimulus in collaborationwith granulocytes. Cytokine and the like are released from activatedgranulocytes and macrophages as well as from injured tissue, andthereby, infiltration of tissue by lymphocytes consisted of T-cells andB-cells which can efficiently perform immune reactions such asproduction of antibody, or repairing the demolished tissue, orregulating the reaction for immune response, is enhanced. Infiltrationby lymphocytes reaches to a peak in dozens of hours after the initialstimulus.

The immune reaction is a quite important action for maintaining thebiosis of an individual. However, since the regulation of the reactionin an individual is imperfect with respect to the purpose of maintainingits biosis, the reaction may sometime cause crucial negative effects.Typical phenomena of such negative effects are development of disordersreferred to as autoimmune diseases. As a group of this kind ofdisorders, for example, atopic dermatitis, rheumatoid arthritis,Behcet's syndrome, systemic lupus erythematosus, ulcerative colitis,Crohn's disease, chronic thyroiditis and other collagen diseases areknown. In the case of inflammations caused, for example, by invasion ofextraneous substance such as infection, or by tissue demolition such asburn injury, the causes of the inflammation may be identified. However,in the case of autoimmune diseases, there is no specific cause factor,or the factor has not been identified. Thus, the autoimmune disease isan intractable disease with unknown cause. However, it is well knownthat the phenomenon commonly observed in this group of disorders isdisease-specific infiltration of the tissue by leukocytes, inparticular, by lymphocytes, monocytes and macrophages.

Thus, in autoimmune diseases and chronic state of inflammation andinfection, lymphocytes and monocytes play universally a central role inthe immune reaction through leukocyte infiltration. Therefore, search ordetection of leukocyte infiltration by lymphocytes, monocytes andneutrophils, and determination of their precise levels are quiteimportant to perform an effective medical treatment, which may keeppatients away from inappropriate medication and relieve mental distressand expense for the treatment, and may contribute to reduce the cost ofhealth insurance.

Thus, since the diagnostic imaging is a useful tool for searching anddetecting leukocyte infiltration and determination its precise level,various radioactive agents and its application have been investigated inthe field of nuclear medicine.

Gallium-67 (⁶⁷Ga) citrate has long been used as an agent forscintigraphy of inflammation (see, for example, Ebright, Jr. et al.,Arch. Int. Med., 142, 246-254 (1982)). However, this agent has nospecificity for infection site or inflammation site in addition, theradiation energy of gamma ray from ⁶⁷Ga is insufficient and not suitablefor obtaining a good photographic image by a common gamma camera.Further, it requires waiting time for about 72 hours from injection ofthe agent to complete imaging.

In the next place, as a method for imaging the infection site by nuclearmedicine, leukocyte labeled with Indium-111 (hereinafter designated as¹¹¹In-leukocyte) was used (see, for example, Peters, A M. et al., J.Nucl. Med., 33, 65-67 (1992)). Thakur et al. have reported on in vitrolabeling of neutrophils with radioactive nuclides, and analyzed widelyand discussed on its utility (Thakur et al., Sem. Nucl. Med., 14, 10-17(1984)). In this method, neutrophils of an individual were labeled invitro with Indium-111 (hereinafter designated as In-111), and thelabeled neutrophils were used for in vitro kinetic studies. Also, thelabeled neutrophils were able to be used for imaging inflammatory focusin an acute phase of an individual.

However, when ¹¹¹In-leukocyte is utilized, preparation of a radioactivelabeled compound requires steps of aseptic withdrawal of autologousblood, aseptic isolation of leukocytes from the blood, aseptic labelingof leukocytes, and back injection of leukocytes labeled with radioactivenuclide to the patient, and takes a considerable time for more than 2hours. Also, it is considered that 12 to 48 hours of waiting period fromback injection of the labeled leukocytes is required to obtain anoptimal pictorial image. Further, radioactivity of 200 μCi per 1×10⁷cells of leukocytes which is normally used for imaging studies ishazardous for leukocytes when In-111 is employed because of its highradioactive energy. Under the above condition, granulocytes mainlycomposed of neutrophils in leukocytes survive the labeling, butlymphocytes and the like die out immediately after labeling (Chianelli,M. et al., Nucl. Med. Comm., 18, 437-455 (1997)). Therefore, it isdifficult to monitor dynamic states of lymphocytes and monocytes usingIn-111 labeled leukocytes. Furthermore, from the viewpoint of the saferadiation dosage, an administration quantity of radioactivity islimited, in many cases resulting in deterioration of pictorial imagequality.

Leukocytes labeled with technetium-99m (Tc-99m) developed in the nextplace are able to cut down the waiting time for imaging which had been aproblem with ¹¹¹In-leukocytes, and able to monitor dynamic states oflymphocytes and monocytes, and further able to administer a considerablylarger quantity of radioactivity compared to In-111 (see, for example,Vorne, M. et al., J. Nucl. Med., 30, 1332-1336 (1989)). However, sincethe labeling stability of Tc-99m is not good enough, accumulation in anon-target metabolic organ becomes a problem accordingly. In theproblems of long preparation time and handling of blood sample, thismethod is similar to those of ¹¹¹In-leukocytes (Chianelli, M. et al.,Nucl. Med. Comm., 18, 437-455 (1997)).

Trial of developing labeled monoclonal and polyclonal antibodies withradioactive nuclides having binding property to human leukocytesincluding monocytes, neutrophils, granulocytes and the like have beencarried out. For example, ^(99m)Tc-labeled anti-granulocyte monoclonalantibody (refer for example, Lind, P. et al., J. Nucl. Med., 31, 417-473(1990)) and ¹¹¹In-labeled non-specific human immunoglobulin (¹¹¹In-HIG,see, for example, LaMuraglia, G M. et al., J. Vasc. Surg., 10, 20-28(1989)) have been studied for detection of inflammation site caused byinfection. However, ¹¹¹In-HIG has similar drawback with respect to thewaiting period of 24 to 48 hours from administration for obtainingoptimal pictorial image.

In addition, ¹¹¹In-HIG has been considered to accumulate in theinflammation site and enables to depict the site (see, for example,Rubin, R. et al., J. Nucl. Med., 29, 651-656 (1988)), however in thisregard, there are two hypothesis on the mechanism of accumulation. Oneidea is that accumulation of ¹¹¹In-HIG in the inflammation site proceedsby binding to Fc-receptor on the surface of leukocytes which infiltrateinto the inflammation site (Fischman, A. et al., J. Nucl. Med., 31,1199-1205 (1990)), and the other is that, besides the leukocyteinfiltration, local diapedesis of ¹¹¹In-HIG from blood vessel occurredby enhanced permeability through blood vessel, which is observedsimilarly for proteins like albumin (Morrel, E. et al., J. Nucl. Med.,30, 1538-1545 (1989)).

Until now, studies on the biomolecules having binding property toleukocytes using other proteins have been reported.

Van der Laken, C J. et al. have reported on labeled interleukin 1 ofinflammatory cytokine with radioactive iodine (van der Laken, C J. etal., European J. Nucl. Med., 22, 1249-1255 (1995)).

Signore et al. have reported on the chronic inflammatory disease usinglabeled interleukin 2 of inflammatory cytokine with radioactive iodine(Signore, A. et al., Nucl. Med. Comm., 13, 713-722 (1992)).

Hay, R V. et al. have reported on the chemically induced inflammationusing labeled interleukin 8 of inflammatory cytokine with radioactiveiodine (Hay, R V. et al., Nucl. Med. Comm., 18, 367-378 (1997)).

These radiolabeled compounds have successfully depicted images of acuteinflammation such as infection, or chronic inflammation such asautoimmune diseases.

However, from the viewpoint that these proteins including antibodieshave large molecular weights, the diapedesis of these proteins withblood components may cause problems in acute inflammation which withenhanced vascular permeability (Roitt, I. et al., Essential Immunology,8^(th) edn. Oxford, Blackwell Scientific (1994)). Molecules with amolecular weight of around 2000 do not remain at the same site ofdiapedesis for long time even if these are leaked out, but proteins likealbumin (molecular weight: about 64000) tend to remain at the same sitecompared to compounds with low molecular weights because of their largemolecular sizes. Threfore, it is difficult to judge whether theaccumulation is inflammation specific or not (Morrel, E. et al., J.Nucl. Med., 30, 1538-1545 (1989)).

Small and easily synthesizable molecules are preferable to be usedroutinely as a radioactive medicinal agent. Peptides labeled withradioactive nuclides are considered suitable as a synthetic compoundwith low molecular weight, which can bind selectively to leukocytes inthe whole blood and can be injected directly into patients, enabling toimage the seat of disease of infection and inflammation by determiningthe site of leukocytes accumulation.

For example, Moyer et al. have reported on the cumulativecharacteristics of Tc-99m-labeled peptide compounds derived from acarboxyl terminal sequence of platelet factor 4 (PF-4) having bindingproperty to multi-sulfated glycans such as heparin (Moyer, B R. et al.,J. Nucl. Med., 37, 673-679 (1996)). This compound (PF-4 peptide-heparin)is a complex of peptide with 23 amino acid residues containing Tc-99mchelated amino acid sequence (molecular weight: about 2600) and heparin(molecular weight: about 7000 to 25000), constructing a single moleculewith molecular weight of about 10000 to 30000.

Heparin bound PF-4 peptide cannot be an agent indicative of onlyaccumulation to the site of leukocyte infiltration because of its largemolecular weight like proteins. Therefore, an agent consisting of acompound with low molecular weight showing little non-specificaccumulation by enhanced capillary permeability which indicates trueinfiltrated leukocytes has been needed. In addition, use of heparin hassometime to be limited because of its physiological activity.

Dahlman, T. et al. and Ringe, J D. et al. have reported on the risk ofside effects of heparin that administration of heparin may inducereduction of bone density and long-term administration may causeosteoporosis (Dahlman, T. et al., Br. J. Obsted Gynaecol., Mar, 97, 3,221-228 (1990); Ringe, J D. et al., Geburtshilfe Frauenheilkd., 52, 7,426-429 (1992)). In addition to this, there may be a risk of sideeffects brought by physiological function of heparin including, forexample, antithrombin activity, inhibition of thromboplastin production,and inhibition of platelet aggregation, or careful treatment is requiredwhen the patient has hemorrhagic disease or a potential to becomehemorrhagic or severe liver disease and kidney disease. As stated above,heparin has many cautious points for its use.

As to other peptides labeled with radioactive nuclides,formyl-methionyl-leucyl-phenylalanyl (fMLF)-containing peptides havebeen reported in the prior art.

Day et al. have reported on the ¹²⁵I labeling of chemotactic formylatedpeptide (fMLF) (Day, A R. et al., FEBS Lett., 77, 291-294 (1977)).

Jiang et al. have reported on in vivo accumulation of ¹²⁵I-labeledchemotactic formylated peptide (fMLF) in inflammation site (see, forexample, Jiang, M S. et al., Nuklearmedizin, 21, 110-113 (1982)).

Fisherman et al. have disclosed ¹¹¹In labeling of chemotactic formylatedpeptide (fMLF) linked through DTPA (see JP 2931097).

Verbeke et al. have reported on Tc-99m labeling of chemotacticformylated peptide (fMLF) linked through mercaptoacetyl-glycyl-glycine(see, for example, Verbeke, K. et al., Nuclear Medicine & Biology, 27,769-779 (2000)).

Baidoo et al. have reported on Tc-99m labeling of chemotactic formylatedpeptide (fMLF) linked through diaminodithiol compound (refer, Baidoo, K.E. et al., Bioconjugate Chemistry, 9, 208-217 (1998)).

Additionally, there are reports on use of the chemotactic formylatedpeptides (fMLF) labeled with radioactive nuclides for in vitro labelingof leukocytes with radioactive nuclides through photo-affinity thereof(see, for example, U.S. Pat. No. 4,986,979).

Also, there are reports on chemotactic formylated peptides (fMLF)capable of being labeled with radioactive nuclides (see, for example,U.S. Pat. No. 5,792,444).

Chemotactic formylated peptide (fMLF) is considered to bind toleukocytes through formylated peptide receptor (hereinafter designatedas receptor FPR) (Niedel, J. E. et al., Science, 205, 4413, 1412-1414(1979)), and the leukocytes expressing receptor FPR are neutrophils andmonocytes (Lewis, S L. et al., Inflammation, 4, 363-375 (1983)).

Normal composition of leukocytes present in human blood is composed ofabout 50% of neutrophils and 10% of monocytes. It has been reported thatmost of leukocytes bound with some analogues of known chemotacticformylated peptides are neutrophils because the population of monocytesin blood is only one fifth or less of neutrophils, and the binding ofthe peptides to lymphocytes and monocytes is not strong (Verbeke, K. etal., Nucl. Med. Biol., 27, 769-779 (2000)).

Also, accumulation of formylated peptide labeled with a radioactivenuclide is observed in acute inflammation such as infectious diseases ofbacteria with neutrophils infiltration (see, Babich, J W. et al., J.Nucl. Med., 34, 2176-2181 (1993)), but there is no report on theaccumulation of said peptide to the disease diagnosed as chronicinflammation.

Further, in clinical diagnosis, diseases having a need of imagediagnosis including nuclear medicine testing or MRI (magnetic resonanceimaging) testing are mostly used in diseases having difficulty inidentifying a lesion by primary diagnosis such as in vitro testing orthe cases of diseases in chronic state. In such case, medical treatmentsfor inhibiting immune reaction and leukocyte infiltration byadministration of steroid drug and leukocytes reducing treatment havefrequently been applied. Therefore, an agent with low molecular weightwhich is unsusceptible to enhanced vascular permeability and whichenables to visualize immune reaction and leukocyte infiltration toperform image diagnosis of the diseases accompanied by an inflammationof immune reaction including chronic inflammation has been needed.

The method for labeling peptides and polypeptides with Tc-99m is wellknown art (see, for example, JP-A-8-231587).

SUMMARY OF THE INVENTION

In consideration of the circumstances of prior art, an object of thepresent invention is to provide a compound having binding propertiesspecific to leukocytes, i.e., neutrophils, monocytes and lymphocytesboth in vivo and in vitro and capable to be labeled with a radioactivemetal or a paramagnetic metal, and a medicinal composition containingsaid compound in labeled state as the active ingredient which is usefulfor SPECT image diagnosis, PET image diagnosis, MRI image diagnosis,radioactive treatment and so on, wherein imaging is performed in a sitewith vigorous leukocyte infiltration accompanied by an immune reactionin an individual.

Namely, the present invention relates to a compound binding toloeucocytes represented by the following formula (1):Z-Y-Leu-Phe-(X)_(n)-Lys(NH₂)_(m)-ε(—R-(T)₁-U)   (1)wherein, in the formula (1), Z represents a protecting group for anamino group; Y represents Met or Nle; in (X)n, X represents a spacerconsisting of one or more of amino acids and/or synthetic organiccompounds, and n represents 1 or 0; in (NH₂)_(m), NH₂ represents anamide group as a protecting group for a carboxyl group in the α positionof Lys, and m represents 1 or 0; in E (—R-(T)₁-U), R represents Ser orThr binding to an ε-amino group of Lys through an amide bond, Trepresents a spacer consisting of one or more of amino acids and/orsynthetic organic compounds, l represents 1 or 0, and U represents agroup which can be labeled with a metal; with the proviso that said Xand T may be the same or different from each other).

Furthermore, the present invention relates to a medicinal compositioncontaining the compound binding to leukocytes represented by the aboveformula (1) in labeled state with a radioactive metal or a paramagneticmetal as the active ingredient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a HPLC chromatogram of Tc-99m-peptide 4.

FIG. 2 shows a HPLC chromatogram of Tc-99m-peptide 6.

FIG. 3 shows a distribution of Tc-99m-peptide in the rabbit blood.

FIG. 4 shows an image of Tc-99m-peptide 3 in a model rabbit withinfectious disease. Left side is an image 2 hours after theadministration; right side is an image 22 hours after theadministration.

FIG. 5 shows an image of Tc-99m-peptide 4 in a model rabbit withinfectious disease. Left side is an image 2 hours after theadministration; right side is an image 22 hours after theadministration.

FIG. 6 shows an image of Tc-99m-peptide 6 in a model rabbit withinfectious disease. Left side is an image 2 hours after theadministration; right side is an image 22 hours after theadministration.

FIG. 7 shows an image of Tc-99m-peptide 8 in a model rabbit withinfectious disease. Left side is an image 2 hours after theadministration; right side is an image 5 hours after the administration.

FIG. 8 shows an image of Tc-99m-peptide 9 in a model rabbit withinfectious disease. Left side is an image 2 hours after theadministration; right side is an image 5 hours after the administration.

FIG. 9 shows an image of Tc-99m-peptide 12 in a model rabbit withinfectious disease. Left side is an image 2 hours after theadministration; right side is an image 22 hours after theadministration.

FIG. 10 shows an image of Tc-99m-peptide 13 in a model rabbit withinfectious disease. Left side is an image 2 hours after theadministration; right side is an image 5 hours after the administration.

FIG. 11 shows an image of Tc-99m-peptide 14 in a model rabbit withinfectious disease. Left side is an image 2 hours after theadministration; right side is an image 5 hours after the administration.

FIG. 12 shows an image of Tc-99m-peptide 15 in a model rabbit withinfectious disease. Left side is an image 2 hours after theadministration; right side is an image 5 hours after the administration.

FIG. 13 shows an image of Tc-99m-peptide 16 in a model rabbit withinfectious disease. Left side is an image 2 hours after theadministration; right side is an image 5 hours after the administration.

FIG. 14 shows an image of Tc-99m-peptide 17 in a model rabbit withinfectious disease. Left side is an image 2 hours after theadministration; right side is an image 5 hours after the administration.

FIG. 15 shows an image of Tc-99m-peptide 18 in a model rabbit withinfectious disease. Left side is an image 2 hours after theadministration; right side is an image 5 hours after the administration.

FIG. 16 shows a time course change of urinary excretion ofTc-99m-peptides in a normal rat.

FIG. 17 shows a time course change of accumulation of Tc-99m-peptides inthe small intestine of a normal rat.

FIG. 18 shows a distribution of Tc-99m-peptide in the human blood.

FIG. 19 shows a distribution of Tc-99m-labeled peptide in the blood of amodel rat with colitis.

FIG. 20 shows an image of Tc-99m-labeled peptide 3 in a model rat withcolitis. Left side is an image 30 minutes after the administration;right side is an image 120 minutes after the administration.

FIG. 21 shows an image of Tc-99m-labeled peptide 6 in a model rat withcolitis. Left side is an image 30 minutes after the administration;right side is an image 120 minutes after the administration.

FIG. 22 shows an image of Tc-99m-labeled leukocytes in a model rat withcolitis. Left side is an image 30 minutes after the administration;right side is an image 120 minutes after the administration.

FIG. 23 shows inflammation/organ ratios of Tc-99m-labeled peptides in amodel rat with colitis.

FIG. 24 shows images of peptide 6 on autoradiography and immuno-stainingof a rat with colitis. Left side is autoradiograph; center isimmuno-staining by anti-granulocytes antibody; right side isimmuno-staining by anti-monocytes antibody.

FIG. 25 shows images of peptide 14 on autoradiography andimmuno-staining of a rat with colitis. Left side is autoradiograph;center is immuno-staining by anti-granulocytes antibody; right side isimmuno-staining by anti-monocytes antibody.

FIG. 26 shows images of Tc-99m-labeled leukocytes on autoradiography andimmuno-staining of a rat with colitis. Left side is autoradiograph;center is immuno-staining by anti-granulocytes antibody; right side isimmuno-staining by anti-monocytes antibody.

FIG. 27 shows inflammation/normal tissue ratios of autoradiography of arat with colitis using Tc-99m-labeled peptide and Tc-99m-labeledleukocytes.

FIG. 28 shows inhibition rates of peptides for binding betweenrecombinant human receptor and [³H]-FMLP.

FIG. 29 shows an image of Tc-99m-labeled peptide 6 in a model rabbitwith infectious disease without inhibition of FMLP. Left side is animage 2 hours after the administration; right-side is an image 5 hoursafter the administration.

FIG. 30 shows an image of Tc-99m-labeled peptide 6 in a model rabbitwith infectious disease with inhibition of FMLP.

BEST MODE FOR CARRYING OUT OF THE INVENTION

Hereinbelow, mode for carrying out of the present invention will bedescribed. All amino acids used in the present specification are denotedby single or three characters expression, and unless otherwise noted,the left hand shows the N-terminal side and the right hand shows theC-terminal side. Inside of the parentheses following an amino acidexpresses, unless otherwise noted, a peptide or an organic compoundbound to the side chain. Also, an amino acid sequence in the parenthesisis expressed in such a manner as the right hand for the N-terminal sideand the left hand for the C-terminal side to make easy for understandingthe whole structure. Further, in the present specification, an aminoacid with D-configuration is designated as D-amino acid.

A compound binding to leukocytes of the present invention is representedby the following formula (1):Z-Y-Leu-Phe-(X)_(n)-Lys(NH₂)_(m)-ε(—R-(T)₁-U)   (1)Namely, said formula (1) comprises a binding site Z-Y-Leu-Phe- to thereceptor FPR of leukocytes; a binding part —R— consisting of Ser or Thrwhich elevates the binding ratio to monocytes and lymphocytes of thewhole leukocytes; a structure —U— which can be labeled with aradioactive metal or a paramagnetic metal; and spacers —(X)_(n)—,-Lys(NH₂)_(m)— and -(T)₁- which bind these groups together.

More specifically, the following compounds binding to leukocytes areexemplified as preferred embodiments.

-   formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-Cys-Gly-Asn);-   formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-Cys-Asp-Asp);-   formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-Cys-Gly-Asp);-   formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-Asp-Cys-Asp-Asp);-   formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic    acid);-   formyl-Nle-Leu-Phe-Lys(NH₂)-ε-(-Ser-D-Ser-Asn-D-Arg-Cys-Asp-Asp);-   formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-diethylenetriamine    pentaacetic acid);-   formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-1,4,8,11-teraazacyclotetradecane-butyric    acid);-   formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-Asp-1,4,8,11-tetraazacyclotetradecane-butyric    acid);-   formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Ser-Asn-1,4,8,11-tetraazacyclotetradecane-butyric    acid);-   acetyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-Asp-Cys-Asp-Asp);-   carbamyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-Asp-Cys-Asp-Asp);    and-   methyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-Asp-Cys-Asp-Asp).

With regard to the compound binding to leukocytes represented by theformula (1), in the binding site to the receptor FPR Z-Y-Leu-Phe-, Z isa protecting group for an amino acid including, for example, acyl groupwith 1 to 9 carbon atoms such as formyl and acetyl groups, an acyloxygroup with 2 to 9 carbon atoms such as t-Boc group, a lower alkyl groupwith 1 to 6 carbon atoms such as methyl, ethyl and propyl groups, andcarbamyl group. When N-terminal of Met or Nle in Y is a formyl group, itshows binding property to the receptor FPR of leukocytes recognizing theformylated peptide, and an acetyl group and a t-Boc group also showbinding property to the receptor as well.

In Z-Y-Leu-Phe- of the above formula (1), Y represents Met or Nle as anamino acid. The receptor FPR, which is one of receptors regularlyexpressed on the cell membrane of neutrophils and monocytes inleukocytes, has a strong binding property to a formylated peptide, andhence shows a binding property to Met. The receptor also shows a bindingproperty to Nle as well which has a similar steric structure to that ofMet.

Leu and Phe have a high binding property to neutrophils and monocytes.The receptor FPR which has a strong binding property to formylatedpeptide also has a binding property to peptides such as formyl-Met,formyl-Met-Met, formyl-Met-Met-Leu and formyl-Met-Leu-Leu, and moststrongly to formyl-Met-Leu-Phe.

The most prominent feature of the present invention is R which is boundto Z-Y-Leu-Phe- in the above formula (1) through the spacer X and anε-amino group of -Lys(NH₂)_(m)—.

R is selected from Ser and Thr which are amino acids having a hydroxylgroup in the side chain. By utilizing this structure having Ser or Thrwhich can be labeled with a metal, remarkable binding property tolymphocytes and monocytes has been realized, which has not been seenwith the conventional leukocytes-binding compounds with low molecularweight. The conventional peptides consisting of only Z-Y-Leu-Phe-, whichis a binding site to the receptor FPR, showed a binding property toneutrophils and monocytes, but hardly showed a binding property tolymphocytes. By combining Z-Y-Leu-Phe- of the binding site to thereceptor FPR and a structure which can be labeled with a metal throughSer or Thr bound to ε-amino group of Lys, binding ratio of the peptideto monocytes and lymphocytes was improved significantly. In consequence,it has been realized that the compound binding to leukocytes of thepresent invention represented by the formula (1) has an ability to bindto all species of leukocytes, namely neutrophils, monocytes andlymphocytes. For example, in the case of the conventionalhemocyte-binding compounds, a ratio of the compound bound to lymphocytesand monocytes to the compound bound to the whole leukocytes is in therange of about 12% to 35%, whereas, in the case of the hemocyte-bindingcompound of the present invention, a ratio of the compound bound tolymphocytes and monocytes to the compound bound to the whole leukocytesis elevated up to the range of about 18% to 65%. This indicates that alesion with vigorous infiltration by leukocytes can be targeted, andthat the compound of the present invention is a useful-agent fordiagnosis or medical treatment of the diseases accompanied by leukocyteinfiltration.

In the compound binding to leukocytes of the present invention, eachdistance between the binding site to the receptor FPR Z-Y-Leu-Phe-, inparticular, Ser or Thr of the binding site R to the receptor oflymphocytes and monocytes, and the structure U to be labeled with aradioactive metal or a paramagnetic metal may be adjusted adequately bylinking these parts through a spacer X, and ε-amino group of-Lys(NH₂)_(m)— and a spacer T. Owing to this, these parts can be linkedeach other while the binding property to the receptor which issignificantly influenced by the steric structure is maintained. Namely,in order to bind the C-terminal of Ser or Thr of R with the C-terminalof Z-Y-Leu-Phe-, it is preferable to add Lys having a side chain with 4carbon atoms and an amino group to the C-terminal group of Z-Y-Leu-Phe-and then Ser or Thr is linked to the ε-amino group of Lys. When furtherspatial distance is needed, the spacer X may be added.

Each of X and T is a spacer consisting of one or more of amino acidsand/or synthetic organic compounds, which may be a component of thecompound binding to leukocytes of the present invention when needed. Xand Y may be the same or different from each other. However, Cys residueis not an appropriate amino acid to use as a component of X or T,because the sulfanyl group may form an intramolecular or anintermolecular disulfide bond resulting in polymeric form such as dimer,and this structural change may influence significantly on bindingproperty to the receptor. Also, Pro is not preferable for binding to thereceptor, because if Pro is contained in X or T, the steric structure isrestricted and degree of spatial freedom is limited accordingly.Therefore, it is desirable that Cys and Pro are excluded from the aminoacids contained constructing the sequence of X or T.

More specifically, an amino-acid to be used for X which has littleinfluence on the binding property to the receptor includes, for example,uncharged amino acids such as Gly, Ala, Val, Leu and Ile, Nle, Tyr, andNle-Tyr. As to an amino acid for T, for the purpose to provide adistance from the binding site to the receptor to the structure to belabeled with a radioactive metal or a paramagnetic metal, or to controlin vivo dynamic state in the living body, or to provide resistanceagainst metabolism in the living body, the same amino acids as describedabove, non-amino acid compounds or the combination thereof may be used.Also, L- and D-form amino acids other than the above amino acids,hydrophobic amino acids such as Gly, and polar and charged amino acids(acidic or basic) such as Arg, Asp, Glu and Lys may be used.

The synthetic organic compound constructing the spacer includes, forexample, compounds such as 1,5-hexadiene, trans-2-methyl-1,3-pentadieneand 4-methyl-3-nitroacetophenone, each having a hydrophobic functionalgroup such as methyl group, ethyl-group and benzyl group; compounds suchas (±)-2-methyl-2,4-pentanediol(hexylene glycol) and3-methyl-1,3,5-pentanetriol having a polar functional group such ashydroxyl group and amid group; compounds such as methylenesuccinic acid,4-maleinimide butyric acid and 6-maleinimide caproic acid having acharged functional group such as carboxyl group, amino group and iminogroup.

With regard to the group U which can be labeled with a metal, a groupconsisting of one or more of amino acids or a group consisting ofnon-amino acid compounds may be used. As the group consisting of one ormore of amino acids, the peptide represented by -Cys-A1-A2 may be used(with the proviso that A1 and A2 are amino acids except for Cys andPro). These peptides include, for example, -Cys-Gly-Asp, -Cys-Asp-Asp,-Cys-Asp-Gly, -Cys-Gly-Glu, -Cys-Glu-Glu, -Cys-Glu-Gly, -Cys-Gly-Asn,-Cys-Asn-Asn, -Cys-Asn-Gly, -Cys-Gly-Gln, -Cys-Gln-Gln, -Cys-Gln-Gly,-Cys-Gly-Lys, -Cys-Lys-Lys, -Cys-Lys-Gly, -Cys-Gly-Arg, -Cys-Arg-Arg and-Cys-Arg-Gly.

The group consisting of non-amino acid compound which can be labeledwith a metal includes, for example, a nitrogen-containing cycliccompound with 8 to 20 carbon atoms such as1,4,7,10-tetraazacyclododecane(Cyclen),1,4,8,11-tetraazacyclotetradecane(Cyclam),1,4,8,12-tetraazacyclopentadecane and1,4,8,11-tetraazacyclotetradecane-5,7-dione(Dioxocycam); anitrogen-containing cyclic carboxylic acid compound with 8 to 20 carbonatoms such as 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraaceticacid (TETA), 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid(DOTA),1,4,8,11-tetraazacyclotetradecane-5,7-dione-N,N′,N″,N′″-tetraaceticacid, 1,4,7,10-tetraazacyclododecane-butyric acid and1,4,8,10-tetraazacyclododecane-butyric acid; a derivative ofnitrogen-containing cyclic carboxylic acid with 8 to 20 carbon atomssuch as 1,4,7,10-tetraazacyclododecane-1-aminoethylcarbamoylmethyl-4,7,10-tris[R,S]-methylacetic acid (DO3MA) and1,4,7,10-tetraazacyclododecane-1,4,7,10-a,a′,a″,a′″-tetramethylaceticacid (DOTMA); and an alkyleneamine carboxylic acid with 4 to 10 carbonatoms such as ethylenediamine tetraacetic acid (EDTA),diethylenetriamine pentaacetic acid (DTPA), triethylenetetraaminehexaacetic acid and ethyleneglycol-(2-aminoethyl)-N,N,N′,N′-tetraaceticacid (EGTA).

When the compound binding to leukocytes of the present invention is usedas a medicinal composition for image diagnoses, depending on the purposeof diagnosis or the lesion to be treated, imaging of the region to bediagnosed may be carried out in short time by virtues of reduction ofunnecessary radiation to the living body and alleviation of theback-ground effect on image diagnosis by smooth excretion of unnecessarymetabolites by controlling dynamic state in vivo. For example, withregard to the amino acids and the like to be used for constructing thespacers X and T, their metabolic transfer can be controlled toward thegastrointestinal tract by using aliphatic amino acids such as Gly, Ala,Ile, Leu and Val, aromatic amino acids such as Phe, Trp and Tyr,sulfur-containing amino acids such as Met, or compounds containing ahydrophobic functional group such as methyl, ethyl and benzyl group. Inorder to control their metabolic transfer toward the urine and thekidney, charged amino acids or compounds having a charged functionalgroup may be used by selecting hydroxyl amino acids such as Ser and Thr,acidic amino acid amides such as Asn and Gln, charged (acidic and basic)amino acids such as Arg, Asp, Glu and Lys, or synthetic organiccompounds containing a polar functional group such as hydroxyl group andamide group, compounds containing a charged functional group such ascarboxyl group, amino group and imino group. In addition, when adistance between the binding site to the receptor and the structure tobe labeled with a metal is needed, one or more of amino acids orsynthetic organic compounds having a straight chain such as alkyl groupmay be used.

With regard to an amino acid contained in the group U which can belabeled with a metal, the same description as above can be addressed.For example, when Asp or Lys is selected, or a compound containingcarboxyl group or amino group is selected, the major excretion rout ofthe final metabolites after administration of the labeled compound witha radioactive metal may be controlled toward the kidney. Also, when ahydrophobic amino acid such as Gly is selected, the major excretion routof the final metabolites may be controlled toward the gastrointestinaltract.

Further, with regard to an amino acid and the like to be used as acomponent of the spacers X and T, in order to provide a resistanceagainst the metabolism in vivo, D-amino acids or artificial amino acidsand non-amino acids may be used. More specifically, the spacerconsisting of D-amino acid includes, for example, amino acid sequencessuch as D-Arg-Asp, Arg-D-Asp, D-Arg-D-Asp, D-Asp-Arg, Asp-D-Arg,D-Asp-D-Arg, Ser-D-Arg, D-Ser-Arg, D-Ser-D-Arg, D-Arg-Ser, Arg-D-Ser,D-Arg-D-Ser, Ser-D-Asp, D-Ser-Asp, D-Ser-D-Asp, D-Asp-Ser, Asp-D-Ser andD-Asp-D-Ser.

The compound binding to leukocytes of the present invention can besynthesized according to the methods described below.

(1) When said compound is consisted of amino acids only, the compoundcan be synthesized according to the methods such as Boc-method andFmoc-method using commonly used automatic peptide synthesizers such asthe automatic peptide synthesizer made by Applied Biosystems (USA). Asynthesized complex may be purified by simultaneously performingdeprotection and cutting off from the bound state to the solid phasecarrier resin, followed by a high performance liquid chromatography(hereinafter designated as HPLC) using a reversed phase column.Otherwise, the said compound may be obtained by a liquid phase peptidesynthesis, or from animal sources and the like.

(2) When said compound contains a non-amino acid, the compound can besynthesized in most cases by the same method as described above. Forexample, said compound can be synthesized as follows. Namely, Lysresidue or a protection derivative thereof is bound to the solid phaseof carrier resin, and a N-terminal thereof is successively bound with anamino acid residue or a protection derivative thereof of X, or acompound serving as a spacer or a protection derivative thereof, Phe ora protection derivative thereof, Leu or a protection derivative thereofand an amino acid or a protection derivative thereof of Y. Then, anε-amino group on a side chain of Lys bound to the solid phase of carrierresin is activated, and Ser or Thr or a protection derivative thereof ofR is bound thereto, followed by further binding with an amino acid or aprotection derivative thereof of spacer T, or a compound serving as aspacer or a protection derivative thereof and a compound U which can bea group to be labeled with a metal or a protection derivative thereof,then cutting off the synthetic compound of the above formula (1) fromthe carrier resin.

The medicinal composition containing a compound in the labeled state asthe active ingredient obtained by labeling a compound binding toleukocytes of the present invention with a radioactive metal or aparamagnetic metal, may be used, for example, as an agent forradioisotope diagnosis or a radioactive therapeutic agent, inparticular, for image diagnosis and treatment of a site of lesion withvigorous leukocyte infiltration accompanied by an immune reaction in anindividual. Namely, in a disease having active lesion with vigorousinfiltration by neutrophils, monocytes or lymphocytes, or more than 2species of leukocyte cells, detection of the site and measurement of alevel of leukocyte accumulation may be performed using a specificdetector.

Diseases accompanied by an immune reaction in an individual include agroup of disorders consisting of infection, inflammation andaterosclerosis in the mammalian body. Namely, said group of disordersinclude viral infection, bacterial infection, fungal infection,protozoan disease, nematode disease, collagen disease and autoimmunediseases other than collagen disease. In Japan, about 80% of hepatitispatients are viral hepatitis caused by viral infection, and others aremostly autoimmune hepatitis, and therefore, a large number ofinflammatory diseases of liver are the disease having infiltration bylymphocytes and monocytes compared with that by nutrophils. In the caseof disease having relatively low level of neutrophil infiltration likethis, it is pointed out that, if the conventional analogues of fMLF areapplied, leukocyte infiltration of the lesion may be underestimatedbecause the binding to lymphocytes and monocytes is weaker compared withthat to neutrophils. However, the compound binding to leukocytes of thepresent invention enables to correctly identify the leukocyteinfiltration of the disease with little neutrophil infiltration such asmost autoimmune diseases and leishmaniasis, because said compound has ahigh binding property not only to neutrophils but also to lymphocytesand monocytes.

A group of disorders showing a quantity of infiltration by lymphocytesand monocytes compared with neutrophils, or a group showing a largepopulation of lymphocytes and monocytes in the infiltrated leukocytesincludes viral infection, protozoan disease, nematode disease, collagendisease and autoimmune diseases other than collagen disease. The presentinvention is considered effective to the group of disorder accompaniedby immune reaction such as leukocyte infiltration, particularlyeffective to the group of disorders showing a quantity of infiltrationby lymphocytes and monocytes compared with neutrophils, or a groupshowing a large population of lymphocytes and monocytes in theinfiltrated leukocytes.

Further, since the compound of the present invention has a specificbinding property to neutrophils as well as described in the prior art,the compound is also effective to a group of disorders showing aquantity of infiltration by neutrophils compared with those bylymphocytes and monocytes, or a group showing a large population ofneutrophils in the infiltrated leukocytes. Such group of disordersincludes, for example, bacterial endocarditis, cardiac infarction,bronchial pneumonia, lobar pneumonia, infiltrative tuberculosis, acutegastritis, pseudomembranous colitis, yersinia infection, ulcerativecolitis, acute appendicitis, acute angiocholitis, cholecystitis,intratublar proliferative glomerulonephritis, infiltrativeglomerulonephritis, acute pyelonephritis, acute salpingitis, acutecervicitis, acute mastadenitis, acute testitis, acute prostatitis,allergic angiitis, acute purulent inflammation, tuberculous megingitis,acute suppurative osteomyelitis, acute lymphadenitis, and tuberculousperiarteritis.

When the compound binding to leukocytes of the present invention is usedas an agent for radioisotope diagnosis, a preferable embodiment of saidcompound is one labeled with radioactive metal suitable for SPECT imagediagnosis such as Tc-99m, In-111, Ga-67, Sn-117m, Sm-153 and Re-186, orwith a radioactive metal suitable for PET image diagnosis such as Cu-64or Ga-68. When the compound of the present invention is used as aradioactive therapeutic agent, a preferred embodiment of said compoundis one labeled with a radioactive metal such as Y-90, Re-186 or Re-188.When the compound of the present invention is used as a contrast mediumfor MRI image diagnosis, a preferred embodiment of said compound is onelabeled with a paramagnetic metal such as Cu, Fe or Gd in a coordinatedstate.

Labeling of the compound binding to leukocytes of the present inventionwith Tc-99m, Re-186 and Re-188 can be performed according to the commonmethod. Namely, the labeled compound can be prepared by dissolving saidcompound in saline or an aqueous buffer solution or the like, followedby addition of a reducing agent such as stannous chloride, then mixingwith a sodium pertechnetate solution or a sodium perrhenate solution.For labeling with Cu, Cu-64, Fe, Mn, Gd or In-111, the labeled compoundbinding to leukocytes of the present invention can be prepared by mixingthe compound with a slightly acidic aqueous solution containing an ionof Cu, Cu-64, Fe, Mn, Gd or In-111. In the case of labeling with Ga-67,Ga-68 or Y-90, the labeled compound binding to leukocytes can beprepared by mixing said compound with a slightly acidic or a slightlyalkaline aqueous solution containing ions of Ga-67, Ga-68 or Y-90.

When the compound labeled with a radioactive metal is used as an agentfor radioisotope diagnosis or a radioactive therapeutic agent, or thecompound labeled with a paramagnetic metal is used as a contrast mediumfor MRI, the labeled compounds prepared according to the above methodmay be used after additional purification by HPLC to remove impuritiesand unreacted ions of pertechnetate ion, perrhenate ion, In-111 ion, Cuion, Cu-64 ion, Ga-67 ion, Ga-68 ion, Fe ion, Mn ion, Gd ion and Y-90ion.

The compound labeled with a radioactive metal or a paramagnetic metalmay be mixed with pharmacologically acceptable additives to prepare anagent for radioisotope diagnosis, a radioactive therapeutic agent, or acontrast medium for MRI. These additives include, for example,pharmacologically acceptable stabilizers such as ascorbic acid andp-aminobenzoic acid, pH adjusters such as aqueous buffer solution,excipients such as D-mannitol, agents useful for amelioratingradiochemical purity such as citric acid, tartaric acid, malonic acid,sodium gluconate and sodium glucoheptonate. Further, the medicinalcomposition can be provided in a form of a kit for on site preparationby mixing these additives and freeze dried, which is particularly usefulas an agent for radioisotope diagnosis of the present invention.

The agent for radioisotope diagnosis, the radioactive therapeutic agentor the contrast medium for MRI containing the compound binding toleukocytes of the present invention in a labeled state with aradioactive metal can be administrated through a commonly usedparenteral route such as intravenous administration. Dosage andradioactivity of the composition which are thought to enable the imagingand medical treatment are determined considering various conditions suchas body weight and age of the patient, suitable radioactive imaginginstrument, MRI measuring instrument and state of the disease.

For human use, the dosage of diagnostic agent containing the compoundlabeled with Tc-99m is in the range of 37 MBq to 1110 MBq, preferably inthe range of 185 MBq to 1110 MBq as a radioactivity of Tc-99m. In thecase of therapeutic agent containing the compound labeled with Re-186 orRe-188, the dosage is in the range of 37 MBq to 18500 MBq, preferably inthe range of 370 MBq to 7400 MBq as a radioactivity. In the case oftherapeutic agent containing the compound labeled with Y-90, the dosageis in the range of 37 MBq to 3700 MBq, preferably in the range of 37 MBqto 1110 MBq as a radioactivity. The dosage of the compound labeled withother radioactive metals is almost the same. The dosage of a diagnosticagent containing the compound labeled with a paramagnetic metal such asGd, Fe, Mn and Cu is variable corresponding to a host to be treated,sensitivity of the MR imaging instrument, target tissue of the imagingexperiment, specific manner of administration and intended efficacy ofthe use. However, a specific medication for a specific patient dependson various factors including activity (induced relaxation) of thespecific reagent to be used, age, body weight, general health condition,sexuality, meal, administration time, excretion speed, combination ofagents, and judgment by a doctor in attendance.

Effective level of dosage of the labeled compound is between about 0.1 μmol/kg body weight and about 1000 μ mol/kg of body weight per day,preferably between about 0.5 μ mol/kg body weight and about 300 μ mol/kgbody weight per day. The representative preparation contains the labeledcompound in an amount of about 1 mM to 1000 mM, preferably about 10 mMto 500 mm.

Hereinbelow, the present invention is described in more detail usingExamples, but the scope of the present invention should not be limitedthereto. The measuring methods for the compounds obtained and reagentsused in Examples are described below.

(1) Gamma counter: The measurement of distribution of the labeledcompound in blood was performed using Auto-Well Gamma Counter(manufactured by ALOKA Corporation, Japan), and the measurement ofdistribution of the labeled compound in a body was performed using NaISingle Channel Analyzer (manufactured by OHYO KOKEN KOGYO CO., LTD.,Japan).

(2) Gamma camera: GMS-550U (manufactured by TOSHIBA MEDICAL SYSTEMSCORPORATION, Japan) was used.

(3) Reversed phase HPLC: Reversed phase column Millipore Puresil 5 μmC18 (4.6×150 mm) (manufactured by Millipore Corporation, USA) was used.

(4) All of the peptide compounds were synthesized by the solid phasepeptide synthesis method.

(5) ^(99m)TcO₄ ⁻: An eluate as a saline solution from ⁹⁹Mo/^(99m)Tcgenerator (NIHON MEDI-PHYSICS CO., LTD., Japan) was used.

(6) All of the reagents used were extra pure grade.

(7) All of the experimental animals were fed for one week under theconditions of light-dark cycle of every 12 hours before use. During theperiod, intake of feed and water was kept free.

EXAMPLES Example 1

Synthesis of Peptides

Following peptides were synthesized by the solid phase peptide synthesisand used in Examples hereinbelow.

Compounds Binding to Leukocytes of the Present Invention

-   Peptide 3: formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-Cys-Gly-Asn);-   Peptide 4: formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-Cys-Asp-Asp);-   Peptide 5: formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-c-(-Ser-Cys-Gly-Asp);-   Peptide 6:    formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-Asp-Cys-Asp-Asp);-   Peptide 7: formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-cyclam    tetracarboxylic acid);-   Peptide 8:    formyl-Nle-Leu-Phe-Lys(NH₂)-ε-(-Ser-D-Ser-Asn-D-Arg-Cys-Asp-Asp);-   Peptide 9: formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-DTPA);-   Peptide 13: formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-Cyclam    butyric acid);-   Peptide 14:    formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-Asp-cyclam butyric    acid);-   Peptide 15:    formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Ser-Asn-cyclam butyric    acid);-   Peptide 16:    acetyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-Asp-Cys-Asp-Asp);-   Peptide 17:    carbamyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-Asp-Cys-Asp-Asp);    and-   Peptide 18:    methyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-Asp-Cys-Asp-Asp).

In the above peptides, cyclam tetracarboxylic acid, DTPA and cyclambutyric acid mean 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraaceticacid, diethylenetriamine pentaacetic acid and1,4,8,11-tetraazacyclotetradecane-butyric acid, respectively.

Control Peptides

-   Peptide 1: formyl-Nle-Leu-Phe-Nle-Tyr-Lys-Glu-Cys;-   Peptide 2: formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Cys-Asn-Asp);-   Peptide 10: formyl-Met-Leu-Phe-Lys-ε-(-Gly-Gly-Cys);-   Peptide 11: formyl-Met-Leu-Phe-Lys-ε-(-Gly-Gly-Ac-S-Bzl); and-   Peptide 12: formyl-Met-Leu-Phe-Lys-ε-(-Gly-Asp-Ac-S-Bzl).    Synthesis of Peptide 3

Using the peptide synthesizer (Model 430A, Applied Biosystems), thepeptide was synthesized under the condition of 0.5 mM scale in MBHAresin (p-methoxy-benzhydrylamine resin hydrochloride, 1%divinylbenzene-polystyrene copolymer) by means of Boc method. The sidechain of Lys residue in the C-terminal was protected with a Fmoc group.After extension of the peptide chain and formylation of the N-terminalamino group, the side chain Fmoc group of the Lys residue was cleft by20% piperidine/DMF to extend the peptide chain toward the side chaindirection. The peptide was clipped off under the reaction in a mixtureof hydrogen fluoride anhydride: p-cresol (80:20) at −2° C. to −5° C. for1.0 hour.

Purification was performed in a liquid chromatography (HPLC) by using acolumn: YMC-Pack ODS-A SH-365-5 (30×250 mm) and eluent A: 0.1%TFA/purified water and eluent B: 0.1% TFA/acetonitrile under theconcentration gradient condition from A to B at the eluting rate of 20ml/min. Main peak fractions were collected and freeze dried to obtainthe objective peptide. Purity of the thus obtained peptide wasdetermined by means of the reversed phase HPLC.

Synthesis could also be performed as well by using a preload resin inplace of MBHA resin.

After the peptide was hydrolyzed in 6 M hydrochloric acid at 110° C. for22 hours, an amino acid composition corresponding to the obtained mainpeak was determined and confirmed to be the objective peptide 3, thenthe peak coinciding with the amino acid composition was freeze dried toobtain the objective peptide 3. Molecular weight of the peptide wasconfirmed to be identical with the theoretical value by massspectrometry (hereinafter designated as ESI-MS) for determiningmolecular weight. Analytical values of the amino acid composition(number of each amino acid in a molecule) of the obtained peptide 3 wereshown hereinbelow. In a parenthesis, theoretical values of the aminoacid composition (number of each amino acid in a molecule) of theobjective peptide were shown. Peptide 3: Asp: (1) 1.02; Ser: (1) 0.93;Gly: (1) 1.03; Tyr: (1) 1.00; Phe: (1) 1.01; Lys: (1) 1.00, NH₃ (2)2.10; Cys: (1) 0.86; Leu: (1)+Nle (2) 2.88

Further, molecular weight of the peptide 3 obtained by ESI-MS is shownhereinbelow. The numerical value in the parenthesis indicates atheoretical value of the molecular weight of the objective peptide.ESI-MS: MW=1183.9 (1184.4)

Synthesis of Other Peptides

Other peptides were synthesized and identified in the similar way. Sincethe peptide 1, peptide 10, peptide 11 and peptide 12-were peptides whichwere not amidated in the C-terminals thereof, these were synthesized bythe similar way as shown in the synthesis of the peptide 3 by using HMPresin (4-hydroxy-methyl-phenoxy methyl-copolystyrene-1% divinyl-benzeneresin) in place of MBHA resin. Analytical values of amino acidcomposition and ESI-MS used for identifying each peptide are shownhereinbelow. For the peptide 1 and the peptide 10, only amino acidcompositions are shown.

-   Peptide 1=Glu:(1) 1.04, Leu:(1) 0.99, Tyr:(1) 0.98, Phe:(1) 0.99,    Lys:(1) 1.00, Cys:(1) 0.97, Nle:(2) 2.03.-   Peptide 2=Asp:(2) 2.00, Leu:(1) 0.86, Tyr:(1) 1.00, Phe:(1) 1.01,    Lys:(1) 1.00, NH₃:(2) 1.94, Cys:(1) 0.88, Nle:(2) 2.10, ESI-MS:    MW=1,155.0 (1,155.4).-   Peptide 4=Asp:(2) 2.00, Ser:(1) 0.90, Tyr:(1) 0.99, Phe:(1) 1.01,    Lys:(1) 1.00, NH₃:(1) 1.10, Leu:(1)+Nle:(2) 2.84, Cys:(1) 0.92,    ESI-MS: MW=1,243.0 (1,243.4).-   Peptide 5=Asp:(1) 1.00, Ser:(1) 0.90, Gly:(1) 1.01, Tyr:(1) 0.97,    Phe:(1) 1.00, Lys:(1) 1.03, NH₃:(1) 1.18, Leu:(1)+Nle:(2) 2.86,    Cys:(1) 0.91, ESI-MS: MW=1,185.1 (1,185.4).-   Peptide 6=Asp:(3) 3.19, Ser:(1) 0.97, Tyr:(1) 0.97, Phe:(1) 0.98,    Lys:(1) 1.00, NH₃:(1)1.20, Leu:(1)+Nle:(2) 2.80, Arg:(1) 1.06,    Cys:(1) 0.92, ESI-MS: MW=1,514.4 (1,514.7).-   Peptide 7=Ser:(1) 0.92, Tyr:(1) 1.00, Phe:(1) 1.02, Lys:(1) 1.00,    NH₃:(1)1.15, Leu:(1)+Nle: (2) 2.89, ESI-MS: MW=1,324.3 (1,324.6).-   Peptide 8=Asp:(3) 2.94, Ser:(2) 1.80, Leu:(1) 1.01, Phe:(1) 1.00,    Lys:(1) 1.01, NH₃:(2) 2.10, Nle:(1) 0.95, Cys:(1) 1.01 ESI-MS:    MW=1,324.1 (1,324.5).-   Peptide 9=Ser:(1) 0.91, Tyr:(1) 1.00, Phe:(1) 0.99, Lys:(1) 1.00,    NH₃:(1) 1.23, Leu:(1)+Nle:(2) 2.87, Arg:(1) 1.00, ESI-MS: MW=1,441.4    (1,441.6).-   Peptide 10=Gly:(2) 1.88, Met:(1) 0.98, Phe:(1) 1.00, Lys:(1) 1.02,    Leu:(1) 1.04.-   Peptide 11=Gly:(2) 1.94, Met:(1) 0.91, Phe:(1) 1.00, Lys:(1) 1.02,    Leu:(1) 1.01, ESI-MS: MW=842.5 (842.4).-   Peptide 12=Asp:(1) 0.94, Gly:(1) 0.92, Met:(1) 0.96, Phe:(1) 1.00,    Lys:(1) 1.01, Leu(1) 1.00, ESI-MS: MW=901.5 (902.1).-   Peptide 13=Ser:(1) 0.92, Tyr:(1) 1.00, Phe:(1) 1.01, Lys:(1) 1.01,    NH₃:(1) 1.11, Leu:(1)+Nle:(2) 2.88, ESI-MS: MW=1,178.3 (1,178.5).-   Peptide 14=Asp:(1) 1.01, Ser:(1) 0.91, Tyr:(1) 1.00, Phe:(1) 1.00,    Lys:(1) 1.01, NH₃:(1) 1.06, Leu:(1)+Nle:(2) 2.89, Arg:(1) 1.00,    ESI-MS: MW=1,449.6 (1,449.8).-   Peptide 15=Asp:(1) 1.01, Ser:(2) 1.77, Tyr:(1) 0.98, Phe:(1) 1.00,    Lys:(1) 1.01, NH₃:(2) 2.09, Leu:(1)+Nle:(2) 2.87, ESI-MS: MW=1,379.5    (1,379.7).-   Peptide 16=Asp:(3) 3.02, Ser:(1) 0.93, Tyr:(1) 1.00, Phe:(1) 1.00,    Lys:(1) 1.00, NH₃:(1) 1.10, Leu:(1)+Nle:(2) 2.86, Arg:(1) 1.01,    Cys:(1) 1.07, ESI-MS: MW=1,528.3 (1,528.7).-   Peptide 17=Asp:(3) 2.95, Ser:(1) 0.91, Tyr:(1) 1.00, Phe:(1) 1.00,    Lys:(1) 1.00, NH₃:(1) 1.79, Leu:(1)+Nle:(2) 2.58, Arg:(1) 1.00,    Cys:(1) 1.00 ESI-MS: MW=1,529.4 (1,529.7).-   Peptide 18=Asp:(3) 2.99, Ser:(1) 0.92, Tyr:(1) 0.88, Phe:(1) 0.97,    Lys:(1) 0.97, NH₃:(1) 1.12, Leu:(1)+Nle:(1) 1.85, Arg:(1) 1.00,    Cys:(1) 1.03, MeNle:(1)0.95 ESI-MS: MW=1,500.5 (1,500.7).

Example 2

Tc-99m Labeling of Peptide 1, Peptide 2, Peptide 3, Peptide 4, Peptide5, Peptide 6, Peptide 7, Peptide 8, Peptide 9, Peptide 10, Peptide 13,Peptide 14, Peptide 15, Peptide 16, Peptide 17 and Peptide 18

(1) Method

A solution of Tc-99m-sodium pertechnetate (hereinafter designated as^(99m)TcO4⁻), 1.1-3.0 GBq, was added into a vial containing a mixture ofglucoheptonic acid 40.3 μmol/300 μl and stannous chloride solution 130nmol/50 μl to make the total volume 1.35 ml. The mixture was reacted atroom temperature for 30 minutes with stirring and occasional tumbling. Apart thereof was collected and the labeling rate of Tc-99m ofTc-99m-glucoheptonic acid was confirmed to be 95% or more.

Each of sixteen peptides obtained in Example 1 was dissolved indimethylformamide (DMF) and a concentration thereof in each solution wasadjusted to 0.25-12.5 nmol/200 μl with ultra pure water, 10 mM phosphatebuffer containing 0.9% NaCl, pH 7.4, (hereinafter designated as PBS) or10 mM carbonate buffer, pH 8.0 (hereinafter designated as CB). To eachsolution was added Tc-99m glucoheptonic acid solution 200 μl, mixed bystirring and reacted at 100° C.-120° C. for 10 minutes. After thelabeling was completed, a part of the reaction mixture was collected andthe labeling rate of Tc-99m was determined by using HPLC. The conditionsof HPLC are as follows. Column: Millipore puresil 5 μm C18 (4.6×150 mm);flow rate: 1 ml/min.; detection wavelength: 220 nm; radioactivitydetector: NaI single channel analyzer; eluent A: 0.1% trifluoroaceticacid (hereinafter designated as TFA)/purified water; eluent B: 0.1%TFA/acetonitrile; and concentration gradient: 0 minutes (20% B) 20minutes (50% B)

(2) Results

In Table 1, labeling rates of 16 species of Tc-99m labeled peptides areshown. Results of peptide 4 and peptide 6 as the representativechromatogram in HPLC analysis of the obtained labeled compounds areshown in FIG. 1 and FIG. 2, respectively. Single labeled product wasrecognized in each peptide. Results of the labeling rates shown in Table1 indicated that high Tc-99m labeling with 80% or more could beperformed. TABLE 1 Labeling rate of Tc-99m labeled peptide Labeledcompound Radiochemical purity (%) Tc-99m-peptide 1 94.4% Tc-99m-peptide2 92.3% Tc-99m-peptide 3 97.6% Tc-99m-peptide 4 99.6% Tc-99m-peptide 5 100% Tc-99m-peptide 6  100% Tc-99m-peptide 7  100% Tc-99m-peptide 898.5% Tc-99m-peptide 9 97.7% Tc-99m-peptide 10 91.7% Tc-99m-peptide 1382.0% Tc-99m-peptide 14 81.0% Tc-99m-peptide 15 96.0% Tc-99m-peptide 1694.5% Tc-99m-peptide 17 97.2% Tc-99m-peptide 18 98.9%

Example 3

Distribution in Rabbit Blood

(1) The peptides which were labeled with Tc-99m in Example 2 (Peptide 3,peptide 4, peptide 6, peptide 8, peptide 9, peptide 12, peptide 13,peptide 14, peptide 15, peptide 16, peptide 17 and peptide 18) werepurified by separating into unlabeled peptides and labeled peptidesusing a reversed phase HPLC under the same conditions of HPLC as inExample 2. Gradient elution was performed under the condition of 20% 50%(0.1% TFA acetonitrile/0.1% TFA water): 0→20 minutes. Subsequently,Percoll density-gradient solution was prepared. To an undiluted Percollsolution (Pharmacia Biotech Inc.) (specific gravity 1.130 g/ml) 90 ml,1.5 M NaCl 10 ml was added to prepare an isotonic solution equal to thephysiological saline. This solution was diluted by adding physiologicalsaline to prepare 1.096, 1.077 and 1.063 g/ml of Percoll solutions. Thethus prepared 1.096, 1.077 and 1.063 g/ml of Percoll solutions, each 1ml, were layered over in a 15 ml tube. It was confirmed to have thedesired density by using density marker beads (red: 1.062; blue: 1.075;orange: 1.087; and green: 1.098). The blood used for the test wascollected from the auricular vein of specific pathogen free (SPF),healthy New Zealand White (NZW) strain rabbit, male, body weight about 2kg.

For examining distribution in the blood of rabbit with infection as anacute inflammation model, the blood of rabbit inflamed withStaphylococcus aureus was used in place of the blood of the healthyrabbit. About 10⁸ viable counts of Staphylococcus aureus were suspendedin physiological saline 1 ml, and the bacterial suspension 100 μl wasadministered intramuscularly into the right calf of New Zealand White(NZW) strain rabbit, male, body weight about 2 kg. The blood wascollected from the auricular vein of the rabbit after about 24 hours andused for the examination.

For examining distribution in the blood of a rabbit with ulcerativecolitis as a chronic inflammantion model, the blood of rabbit inflamedwith 2,4,6-trinitrobenzenesulfonic acid (TNBS) was used in place of theblood of the healthy rabbit. A model rabbit of ulcerative colitis wasprepared according to the method of Anthony et al. (Anthony et al. Int.J. Exp. Path., 76, 215-224, 1995). TNBS 360 mg was dissolved in ultrapure water 4 ml and ethanol 3.2 ml was added thereto to prepare 50.0mg/ml 46% ethanol/physiological saline. A tube was inserted per anum inabout 15 cm depth into the intestine of nembutalized New Zealand White(NZW) strain rabbit, 7 weeks age, weighed 1.3-1.4 kg, male, fastedbefore one day, and air 3 ml was infused. A solution of TNBS/46%ethanol/physiological saline 0.8 ml was infused subsequently andmassaged and tilted the posture for 2 minutes. After 4-5 days, the bloodwas collected from the auricular vein of the rabbit and used for theexamination.

The blood of rabbit, 2 ml each, was warmed at 37° C. for 5 minutes in awarm bath. Each sample of four Tc-99m-peptides 3 μl (111 MBq/ml,Tc-99m-peptide 1.8×10⁻¹¹ mol/ml) purified by HPLC was added thereto andincubated for 30 minutes. The blood sample was Percoll density gradientsolution. The layered sample was centrifuged at 2000 rpm (800×g) for 15minutes. After the centrifugation, the tube was frozen and each fractionwas cut off by using a cutter, then the radioactivity of each fractionwas measured using Auto-Well Gamma Counter to determine theradioactivity distribution of four types of Tc-99m-peptide to the eachblood component.

(2) Results

According to the leukocyte counts 1000-8000 cells/μl from thehematological parameter of rabbits and the number of receptor FPR,100,000-120,000/cell, found in the reference, estimated numbers of thereceptor FPR in the blood of rabbits were calculated as 0.17-1.6×10⁻¹²mol/ml, and ratios of peptide/receptor in rabbits were 0.01-0.11.Results showing percentages of radioactivity in each blood component tothe radioactivity in the whole blood are shown in FIG. 3. Resultsshowing percentages of radioactivity of granulocyte fraction to theradioactivity in the total leukocytes, and a radioactivity of lymphocyteand monocyte fraction for the radioactivity in the total leukocytes areshown in Table 2 and Table 3.

In the healthy SPF rabbit blood, distributions of Tc-99m-peptide 3 andTc-99m-peptide 12 in the granulocyte fraction and in the lymphocyte andmonocyte fraction were 5% or less of the radioactivity in the wholeblood and no strong binding was observed.

In the blood of rabbits with infectious disease caused by Staphylococcusaureus, a radioactivity distribution of Tc-99m-peptide 4 in thegranulocyte fraction was 10.78% of the radioactivity in the whole bloodand that of Tc-99m-peptide 4 in the lymphocyte and monocyte fraction was10.22% of the radioactivity in the whole blood after the incubation for30 minutes. The radioactivity of the granulocyte fraction was 50.22% forthe radioactivity of the whole leukocyte, and the radioactivity of thelymphocyte and monocyte fraction was 49.78% for the radioactivity of thewhole leukocyte. A radioactivity distribution of Tc-99m-peptide 6 in thegranulocyte fraction was 18.27% of the radioactivity in the whole bloodand that of Tc-99m-peptide 6 in the lymphocyte and monocyte fraction was20.21% of the radioactivity in the whole blood after the incubation for30 minutes. The radioactivity of the granulocyte fraction was 47.65% forthe radioactivity of the whole leukocyte, and the radioactivity of thelymphocyte and monocyte fraction was 52.35% for the radioactivity of thewhole leukocyte. In the Tc-99m labeled compounds of peptide 8, peptide9, peptide 13, peptide 14, peptide 15, peptide 16, peptide 17 andpeptide 18, 10% or more of the radioactivity of the whole blood weredistributed in the leukocyte, and about 27% to about 77% of theradioactivity of the whole blood were distributed in the lymphocyte andmonocyte fraction.

A radioactivity distribution of the control Tc-99m-peptide 12 in thegranulocyte fraction was 39.73% of the radioactivity in the whole bloodand that of Tc-99m-peptide 12 in the lymphocyte and monocyte fractionwas 8.89% of the radioactivity in the whole blood after the incubationfor 30 minutes. The radioactivity of the granulocyte fraction was 81.58%for the radioactivity of the whole leukocyte, and the radioactivity ofthe lymphocyte and monocyte fraction was 18.42% for the radioactivity ofthe whole leukocyte. From these results, it has become apparent that thedistribution of Tc-99m labeled compounds of peptide 4, peptide 6,peptide 8, peptide 9, peptide 13, peptide 14, peptide 15, peptide 16,peptide 17 and peptide 18 as a part of the present invention in thelymphocyte and monocyte fraction was larger than that of theconventional peptide, Tc-99m-peptide 12, in the blood of rabbits withinfectious disease caused by Staphylococcus aureus.

In the blood of ulcerative colitis model rabbits prepared by TNBS, asshown in FIG. 3, a radioactivity distribution of Tc-99m-peptide 3 in thegranulocyte fraction was 18.44% of the radioactivity in the whole bloodand that of Tc-99m-peptide 3 in the lymphocyte and monocyte fraction was15.94% of the radioactivity in the whole blood after the incubation for30 minutes. The radioactivity of the granulocyte fraction was 53.72% forthe radioactivity of the whole leukocyte, and the radioactivity of thelymphocyte and monocyte fraction was 46.28% for the radioactivity of thewhole leukocyte as shown in Table 3. A radioactivity distribution ofTc-99m-peptide 6 in the granulocyte fraction was 45.44% of theradioactivity in the whole blood and that of Tc-99m-peptide 6 in thelymphocyte and monocyte fraction was 12.60% of the radioactivity in thewhole blood after the incubation for 30 minutes. The radioactivity ofthe granulocyte fraction was 78.27% for the radioactivity of the wholeleukocyte, and the radioactivity of the lymphocyte and monocyte fractionwas 21.73% for the radioactivity of the whole leukocyte. A radioactivitydistribution of the control Tc-99m-peptide 12 in the granulocytefraction was 15.10% of the radioactivity in the whole blood and that ofTc-99m-peptide 12 in the lymphocyte and monocyte fraction was 8.34% ofthe radioactivity in the whole blood after the incubation for 30minutes. The radioactivity of the granulocyte fraction was 64.66% forthe radioactivity of the whole leukocyte, and the radioactivity of thelymphocyte and monocyte fraction was 35.34% for the radioactivity of thewhole leukocyte.

From these results, it has become apparent that the distributions ofTc-99m-peptide 3 and Tc-99m-peptide 6 as a part of the present inventionin the lymphocyte and monocyte fraction were larger than that of theconventional peptide, Tc-99m-peptide 12, in the blood of ulcerativecolitis model rabbits.

From the above, it was shown that the peptide of the present inventionwas bound more strongly with the lymphocytes and the monocytes than withthe granulocytes as compared with the conventional Tc-99m-peptide 12,and it was confirmed that the peptide of the present invention waseffective for treatment of chronic inflammation frequently infiltratedlymphocytes and monocytes. TABLE 2 Binding rate of Tc-99m labeledpeptide for leukocytes of rabbits (Binding rate (%) for wholeleukocytes, n = 3, mean ± SD) Infectious disease model Lymphocytes andLabeled compound Granulocytes monocytes Tc-99m-peptide 4  50.22 ± 10.31 49.78 ± 10.31 Tc-99m-peptide 6 47.65 ± 2.47 52.35 ± 2.47 Tc-99m-peptide8 51.41 ± 6.39 48.59 ± 6.39 Tc-99m-peptide 9 58.51 ± 2.90 41.49 ± 2.90Tc-99m-peptide 12 81.58 ± 3.58 18.42 ± 3.58 Tc-99m-peptide 13 33.45 ±3.69 66.55 ± 3.69 Tc-99m-peptide 14 58.77 ± 5.64 41.23 ± 5.64Tc-99m-peptide 15 58.94 ± 6.47 41.06 ± 6.47 Tc-99m-peptide 16 23.28 ±0.73 76.72 ± 0.73 Tc-99m-peptide 17 50.98 ± 0.44 49.02 ± 0.44Tc-99m-peptide 18 72.35 ± 4.24 27.65 ± 4.24

TABLE 3 Binding rate of Tc-99m labeled peptide for leukocytes of rabbits(Binding rate (%) for whole leukocytes, n = 3, mean ± SD) Ulcerativecolitis model Lymphocytes and Labeled compound Granulocytes monocytesTc-99m-peptide 3  53.72 ± 10.74  46.28 ± 10.74 Tc-99m-peptide 6 78.27 ±3.16 21.73 ± 3.16 Tc-99m-peptide 12 64.66 ± 4.05 35.34 ± 4.05

Example 4

Imaging of Tc-99m Labeled Compounds of Peptide 3, Peptide 4, Peptide 5and Peptide 12 on Rabbit Infectious Disease Model, and Effectiveness onAcute Phase and Subacute Phase Inflammation

(1) Method

Viable Staphylococcus aureus, about 10⁸ counts, were suspended inphysiological saline 1 ml, and the suspension 100 μl was administeredintramuscularly into the right calf of the rabbit, New Zealand Whitestrain (NZW), about 2 kg. After 24 hours, model rabbits which exhibitedapparent inflammation were anesthetized with pentobarbital, and each ofpeptide 3, peptide 4, peptide 5, peptide 6, peptide 7, peptide 8,peptide 9, peptide 12, peptide 13, peptide 14, peptide 15, peptide 16,peptide 17 and peptide 18, which were labeled with Tc-99m, 37-74 MBqeach, was administered to the ear vein. After 5 minutes, 1 hour, 2hours, 3 hours, 4 hours, 5 hours and 22 hours from the administration,images were recorded by using a gamma camera. The time points from 5minutes to 5 hours after the administration are the times from about 24hours to 29 hours after initiating inflammation and corresponds to acutephase inflammation. The time point after 22 hours is the time afterabout 46 hours from initiating inflammation and corresponds to subacutephase inflammation.

(2) Results

Representative figures of obtained results are shown in FIG. 4, FIG. 5,FIG. 6, FIG. 7, FIG. 8, FIG. 9, FIG. 10, FIG. 11, FIG. 12, FIG. 13, FIG.14 and FIG. 15. Regions of interest are set on the images, and ratios ofcounts in the region of interest of 1000 pixel for whole body counts (%injection dose (ID)/K pixel) are shown in Table 4. Ratios indicating[inflammation]/[normal muscle] (ratios of [A]/[M]) determined from theabove ratios are shown in Table 5.

In the prior known Tc-99m-peptide 12, the ratio of [A]/[M] after 2 hoursfrom the administration (26 hours after initiating inflammation, acutephase inflammation) was 10.34±3.34 (mean±standard error) (n=3), and theratio of [A]/[M] after 22 hours from the administration (46 hours afterinitiating inflammation, subacute phase inflammation) was 33.94±20.76(n=3), while the accumulation to inflammatory region was decreased from1.66±0.63% ID/K pixel after 2 hours from the administration (26 hoursafter initiating inflammation) to 0.90±0.29% ID/K pixel after 22 hoursfrom the administration (46 hours after initiating inflammation).

In Tc-99m-peptide 3 of the present invention, the ratio of [A]/[M] after2 hours from the administration (26 hours after initiating inflammation)was 6.55±2.06 (n=5), and the ratio of [A]/[M] after 22 hours from theadministration (46 hours after initiating inflammation) was 54.16±32.86(n=5), while the accumulation to inflammatory region increased from 0.93±0.31% ID/K pixel after 2 hours from the administration (26 hours afterinitiating inflammation) to 3.70±2.67% ID/K pixel after 22 hours fromthe administration (46 hours after initiating inflammation). InTc-99m-peptide 4, the ratio of [A]/[M] after 2 hours from theadministration (26 hours after initiating inflammation) was 6.75±2.71(n=3), and the ratio of [A]/[M] after 22 hours from the administration(46 hours after initiating inflammation) was 29.07±19.97 (n=3), whichwere lower values than those of the prior known Tc-99m-peptide 12, whilethe accumulation to inflammatory region was increased from 1.09±0.22%ID/K pixel after 2 hours from the administration (26 hours afterinitiating inflammation) to 1.85±0.34% ID/K pixel after 22 hours fromthe administration (46 hours after initiating inflammation). InTc-99m-peptide 6, the ratio of [A]/[M] after 2 hours from theadministration (26 hours after initiating inflammation) was 14.25±0.31(n=3), and the ratio of [A]/[M] after 22 hours from the administration(46 hours after initiating inflammation) was 43.84±12.58 (n=3), whilethe accumulation to inflammatory region was increased from 1.22±0.05%ID/K pixel after 2 hours from the administration (26 hours afterinitiating inflammation) to 1.77±0.07% ID/K pixel after 22 hours fromthe administration (46 hours after initiating inflammation).

Generally, most of leukocytes infiltrated into inflammatory region afterabout 24 hours from infection consist of mainly neutrophils (occupyingmostly in granulocytes), thereafter these decrease gradually andmajority of infiltrated leukocytes are changed to monocytes includingmacrophage and lymphocyte. From the clinical standpoint, mostinflammations, which require a nuclear medical test, are inflammationsafter the subacute phase exhibiting significant infiltration ofmonocytes and lymphocytes. From the above results, it was shown that thepeptides of the present invention are extremely useful for the diagnosisnot only in the acute phase inflammation after 26 hours from onset ofinflammation (2 hours after administration) but also in the subacutephase inflammation after 46 hours from onset of inflammation (22 hoursafter administration). TABLE 4 Accumulation of Tc-99m labeled peptide ininflammation on rabbit infectious disease model (% ID/K pixel) (n = 3,Tc-99m-peptide 3: n = 5, mean ± standard deviation) Labeled Elapse oftime after administration compound 5 min. 1 hr 2 hrs 3 hrs 4 hrs 5 hrs22 hrs Tc-99m- 0.39 ± 0.14 0.66 ± 0.18 0.93 ± 0.31 1.08 ± 0.31 1.39 ±0.55 1.63 ± 0.72 3.70 ± 2.67 peptide 3 Tc-99m- 0.95 ± 0.24 0.91 ± 0.141.09 ± 0.22 1.52 ± 0.27 1.76 ± 0.39 1.84 ± 0.27 1.85 ± 0.34 peptide 4Tc-99m- 0.93 ± 0.10 0.94 ± 0.10 1.22 ± 0.05 1.49 ± 0.14 1.59 ± 0.09 1.64± 0.08 1.77 ± 0.07 peptide 6 Tc-99m- 1.59 ± 0.38 0.97 ± 0.34 0.73 ± 0.160.60 ± 0.10 0.60 ± 0.14 0.62 ± 0.11 not peptide 8 conducted Tc-99m- 1.39± 0.22 0.65 ± 0.11 0.47 ± 0.10 0.41 ± 0.10 0.42 ± 0.06 0.43 ± 0.03 notpeptide 9 conducted Tc-99m- 0.86 ± 0.14 1.31 ± 0.38 1.66 ± 0.63 1.62 ±0.63 1.64 ± 0.66 1.59 ± 0.71 0.90 ± 0.29 peptide 12 Tc-99m- 1.40 ± 0.371.09 ± 0.23 0.68 ± 0.12 0.52 ± 0.10 0.54 ± 0.07 0.54 ± 0.08 not peptideconducted 13 Tc-99m- 1.30 ± 0.20 0.82 ± 0.18 0.58 ± 0.04 0.44 ± 0.030.49 ± 0.00 0.51 ± 0.02 not peptide conducted 14 Tc-99m- 1.36 ± 0.300.83 ± 0.02 0.76 ± 0.07 0.81 ± 0.32 0.90 ± 0.33 1.04 ± 0.28 not peptideconducted 15 Tc-99m- 2.93 ± 0.21 0.85 ± 0.21 0.38 ± 0.05 0.23 ± 0.050.16 ± 0.06 0.13 ± 0.03 not peptide conducted 16 Tc-99m- 2.59 ± 0.701.13 ± 0.07 0.62 ± 0.03 0.47 ± 0.04 0.38 ± 0.07 0.36 ± 0.12 not peptideconducted 17 Tc-99m- 2.14 ± 0.16 0.75 ± 0.26 0.34 ± 0.09 0.19 ± 0.110.14 ± 0.06 0.13 ± 0.04 not peptide conducted 18

TABLE 5 A ratio of inflammation/muscle of Tc-99m labeled peptide onrabbit infectious disease model (n = 3, Tc-99m-peptide 3: n = 5, mean ±standard deviation) Labeled Elapse of time after administration compound5 min. 1 hr 2 hrs 3 hrs 4 hrs 5 hrs 22 hrs Tc-99m- 1.64 ± 0.57 3.56 ±1.03 6.55 ± 2.06  8.68 ± 2.89 11.48 ± 2.88 13.11 ± 3.37 54.16 ± 32.86peptide 3 Tc-99m- 1.94 ± 0.49 4.08 ± 1.34 6.75 ± 2.71 10.65 ± 5.53 13.02± 5.76 14.81 ± 6.77 29.07 ± 19.97 peptide 4 Tc-99m- 2.08 ± 0.46 6.16 ±0.59 14.25 ± 0.31  24.31 ± 4.55 30.20 ± 7.49 29.67 ± 7.66 43.84 ± 12.58peptide 6 Tc-99m- 1.31 ± 0.56 4.27 ± 2.15 9.22 ± 3.81 13.33 ± 5.30 18.51± 1.88 19.30 ± 2.18 not peptide 8 conducted Tc-99m- 1.83 ± 0.38 3.91 ±0.52 5.30 ± 2.08  5.79 ± 1.48  8.87 ± 3.02  8.84 ± 3.03 not peptide 9conducted Tc-99m- 2.96 ± 0.63 7.06 ± 1.43 10.34 ± 3.34  13.81 ± 2.9416.88 ± 2.80 19.64 ± 1.24 33.94 ± 20.76 peptide 12 Tc-99m- 1.60 ± 0.222.84 ± 0.46 6.21 ± 1.25  5.60 ± 2.00  9.25 ± 1.63 11.26 ± 2.77 notpeptide conducted 13 Tc-99m- 1.63 ± 0.21 3.88 ± 0.88 12.23 ± 3.30  11.20± 3.73 18.93 ± 3.08 27.79 ± 9.32 not peptide conducted 14 Tc-99m- 2.41 ±0.09 5.94 ± 1.82 8.84 ± 4.41 11.86 ± 6.02 12.88 ± 9.40 13.32 ± 8.76 notpeptide conducted 15 Tc-99m- 2.10 ± 0.31 2.67 ± 0.49 4.02 ± 1.27  5.52 ±0.13  4.14 ± 0.83  4.56 ± 0.53 not peptide conducted 16 Tc-99m- 2.02 ±0.36 3.16 ± 0.97 4.63 ± 1.90  7.29 ± 4.74  7.80 ± 6.33 10.71 ± 0.32 notpeptide conducted 17 Tc-99m- 1.58 ± 0.01 2.21 ± 0.98 3.76 ± 0.11  3.60 ±2.71  4.18 ± 3.09  4.20 ± 2.67 not peptide conducted 18

Example 5

Pharmakokinetics of Tc-99m Labeled Peptide 3, Peptide 4, Peptide 6 andPeptide 12

(1) Method

Biodistributions of four types of Tc-99m labeled compounds consisting ofTc-99m-peptide 3, Tc-99m-peptide 4, Tc-99m-peptide 6 and Tc-99m-peptide12 obtained in Example 2 were examined in normal rats. TheBiodistribution experiments were conducted according to the conventionalmethod for the person skilled in the art. A sample, 3.0-3.7 MBq each,was administered to the tail vein of non-fasting SD (Sprague-Dawley)strain rats (body weight 140-200 g) under anesthesia with ravonal. After5 minutes, 30 minutes, 60 minutes and 180 minutes from theadministration, rats were exsanguinated from the abdominal aorta.Radioactive counts of each extirpated organ to injection dose weremeasured with a NaI single channel analyzer. Weight of each organ wasmeasured for calculation of the biodistribution. A ratio of theradioactivity of each organ to injection dose was shown by a value perorgan (% ID/organ) or a value per gram of organ (% ID/g organ).

(2) Results

Results are shown in Table 6, Table 7, Table 8 and Table 9. Time coursechanges in urine and the small intestine are shown in FIG. 16 and FIG.17 from the results of Table 6, Table 7, Table 8 and Table 9.

According to the results shown in Table 6, Table 7, Table 8, Table 9,FIG. 16 and FIG. 17, it was found that the biodistributions of Tc-99mlabeled peptides in normal rats were greatly different from each otherdepending on the amino acid residues of Z and W of the formula describedin claim 1. Namely, a metabolic pathway of the conventionalTc-99m-peptide 12 might be mainly through the hepatobiliary excretionroute, because of high accumulation in the liver after 5 minutes fromthe administration, high accumulation in the stomach at each time pointas compared with other peptides, high accumulation at each time point inthe small intestine, and high accumulation at 180 minutes from theintestine to the appendix (FIG. 17). Further, it is difficult tovisualize abdominal inflammation such as inflammatory bowel diseasebecause of high accumulation in the small intestine.

Contrary to that, among the peptides of the present invention, Tc-99mlabeled compounds of peptide 3, peptide 4 and peptide 6, in which T andU of the formula described in claim 1 were converted to or added with ahydrophilic amino acid such as a charged amino acid and an acidic aminoacid, were different from the biodistribution of Tc-99m-peptide 12 andwere stimulated for excretion to urine (FIG. 16). In particular, thetendency was significant for Tc-99m-peptide 6. This characteristic isquite important for visualization of abdominal inflammation such asinflammatory bowel disease. Consequently, the peptides of the presentinvention were suggested to be effective for observing the region withabdominal leukocyte infiltration due to low distribution in theabdominal region particularly in the small intestine as compared withthe conventionally known peptide 12. TABLE 6 Biodistribution ofTc-99m-peptide 3 in normal rats Upper column: % ID/organ; Lower column:% ID/g (n = 3, mean ± standard deviation) Organs 5 min. 30 min. 60 min.180 min. Blood 5.345 ± 0.824 1.759 ± 0.769 0.657 ± 0.150 0.318 ± 0.225(0.719 ± 0.067) (0.212 ± 0.073) (0.082 ± 0.008) (0.038 ± 0.025) Heart0.182 ± 0.014 0.054 ± 0.012 0.020 ± 0.003 0.004 ± 0.000 (0.315 ± 0.014)(0.094 ± 0.024) (0.034 ± 0.002) (0.007 ± 0.000) Lungs 0.805 ± 0.1500.395 ± 0.186 0.117 ± 0.011 0.040 ± 0.005 (0.868 ± 0.116) (0.383 ±0.155) (0.128 ± 0.010) (0.045 ± 0.007) Liver 21.912 ± 0.529  16.435 ±3.821  7.339 ± 0.595 1.239 ± 0.132 (3.287 ± 0.215) (2.475 ± 0.543)(1.105 ± 0.089) (0.206 ± 0.026) Spleen 0.169 ± 0.012 0.102 ± 0.003 0.065± 0.013 0.042 ± 0.004 (0.471 ± 0.022) (0.239 ± 0.020) (0.155 ± 0.026)(0.102 ± 0.014) Kidneys 15.350 ± 1.803  19.142 ± 1.543  14.223 ± 1.091 6.016 ± 1.089 (11.297 ± 1.970)  (13.953 ± 2.124)  (10.679 ± 0.785) (4.508 ± 1.013) Stomach 0.500 ± 0.060 0.354 ± 0.196 0.160 ± 0.177 0.058± 0.049 (0.131 ± 0.031) (0.090 ± 0.037) (0.044 ± 0.043) (0.018 ± 0.012)Small 21.307 ± 1.479  33.762 ± 5.276  37.742 ± 2.889  40.273 ± 1.263 intestine (2.691 ± 0.329) (4.504 ± 0.939) (5.165 ± 0.506) (5.787 ±0.208) Appendix 0.431 ± 0.048 0.125 ± 0.035 0.057 ± 0.013 1.636 ± 0.993(0.086 ± 0.010) (0.024 ± 0.008) (0.013 ± 0.004) (0.308 ± 0.171) Colon0.131 ± 0.052 0.050 ± 0.024 0.017 ± 0.006 0.022 ± 0.026 (0.335 ± 0.030)(0.117 ± 0.016) (0.040 ± 0.001) (0.049 ± 0.060) Rectum 0.486 ± 0.0590.164 ± 0.010 0.063 ± 0.005 0.050 ± 0.020 (0.465 ± 0.042) (0.176 ±0.053) (0.062 ± 0.004) (0.051 ± 0.018) Adrenal 0.024 ± 0.005 0.008 ±0.001 0.004 ± 0.002 0.001 ± 0.000 (0.605 ± 0.131) (0.194 ± 0.024) (0.095± 0.043) (0.001 ± 0.000) Ovaries 0.059 ± 0.003 0.020 ± 0.003 0.008 ±0.002 0.001 ± 0.002 (0.735 ± 0.034) (0.255 ± 0.034) (0.106 ± 0.019)(0.013 ± 0.022) Bones of 0.408 ± 0.042 0.165 ± 0.035 0.073 ± 0.008 0.035± 0.006 lower limb (0.332 ± 0.035) (0.139 ± 0.030) (0.060 ± 0.009)(0.029 ± 0.006) Skin 1.033 ± 0.405 0.362 ± 0.067 0.114 ± 0.037 0.026 ±0.010 (0.371 ± 0.019) (0.154 ± 0.046) (0.052 ± 0.004) (0.011 ± 0.001)Muscle 1.115 ± 0.236 0.366 ± 0.115 0.110 ± 0.021 0.026 ± 0.006 (0.146 ±0.018) (0.047 ± 0.013) (0.016 ± 0.002) (0.004 ± 0.001) Urine 0.230 ±0.126 15.575 ± 3.638  34.936 ± 2.443  48.414 ± 2.048  Feces 0.025 ±0.013 0.014 ± 0.009 0.012 ± 0.013 0.618 ± 1.032 Carcas 30.489 ± 1.877 11.147 ± 2.812  4.283 ± 0.300 1.183 ± 0.362 (0.265 ± 0.011) (0.099 ±0.027) (0.037 ± 0.002) (0.010 ± 0.003)

TABLE 7 Biodistribution of Tc-99m-peptide 4 in normal rats Upper column:% ID/organ; Lower column: % ID/g (n = 3, mean ± standard deviation)Organs 5 min. 30 min. 60 min. 180 min. Blood 6.189 ± 1.270 1.922 ± 0.5600.823 ± 0.350 0.126 ± 0.021 (0.846 ± 0.167) (0.252 ± 0.022) (0.109 ±0.031) (0.017 ± 0.003) Heart 0.163 ± 0.036 0.043 ± 0.006 0.026 ± 0.0130.004 ± 0.001 (0.306 ± 0.077) (0.086 ± 0.010) (0.039 ± 0.010) (0.007 ±0.001) Lungs 0.652 ± 0.096 0.243 ± 0.027 0.147 ± 0.016 0.068 ± 0.017(0.726 ± 0.107) (0.279 ± 0.057) (0.160 ± 0.021) (0.075 ± 0.023) Liver18.159 ± 2.621  11.820 ± 1.257  6.583 ± 0.266 1.358 ± 0.284 (2.810 ±0.295) (1.889 ± 0.186) (1.032 ± 0.061) (0.232 ± 0.065) Spleen 0.144 ±0.010 0.110 ± 0.014 0.076 ± 0.015 0.059 ± 0.009 (0.367 ± 0.064) (0.275 ±0.032) (0.192 ± 0.031) (0.160 ± 0.027) Kidneys 5.643 ± 2.131 4.671 ±0.594 3.938 ± 0.323 3.433 ± 1.054 (4.354 ± 1.312) (3.806 ± 0.604) (3.365± 0.433) (2.814 ± 0.926) Stomach 4.484 ± 4.613 0.340 ± 0.145 0.391 ±0.293 0.178 ± 0.092 (0.905 ± 0.747) (0.115 ± 0.042) (0.118 ± 0.085)(0.050 ± 0.025) Small 28.887 ± 3.569  41.173 ± 0.337  44.035 ± 4.359 13.553 ± 1.591  intestine (3.862 ± 0.976) (5.648 ± 0.657) (5.876 ±0.659) (1.997 ± 0.239) Appendix 0.358 ± 0.029 0.116 ± 0.011 0.077 ±0.032 36.011 ± 2.291  (0.089 ± 0.019) (0.029 ± 0.003) (0.018 ± 0.004)(6.306 ± 0.610) Colon 0.244 ± 0.120 0.072 ± 0.024 0.032 ± 0.016 0.115 ±0.014 (0.395 ± 0.046) (0.172 ± 0.089) (0.071 ± 0.031) (0.289 ± 0.115)Rectum 0.476 ± 0.241 0.125 ± 0.004 0.083 ± 0.037 0.060 ± 0.022 (0.565 ±0.124) (0.166 ± 0.019) (0.100 ± 0.057) (0.061 ± 0.013) Adrenal 0.024 ±0.003 0.010 ± 0.001 0.004 ± 0.001 0.001 ± 0.001 (0.604 ± 0.253) (0.242 ±0.067) (0.092 ± 0.009) (0.037 ± 0.014) Ovaries 0.069 ± 0.026 0.016 ±0.005 0.008 ± 0.001 0.003 ± 0.001 (0.861 ± 0.327) (0.204 ± 0.062) (0.101± 0.011) (0.035 ± 0.017) Bones of 0.342 ± 0.017 0.167 ± 0.020 0.0160 ±0.026  0.044 ± 0.015 lower limb (0.304 ± 0.019) (0.150 ± 0.022) (0.095 ±0.002) (0.039 ± 0.012) Skin 0.506 ± 0.217 0.414 ± 0.189 0.099 ± 0.0170.022 ± 0.006 (0.321 ± 0.039) (0.168 ± 0.016) (0.062 ± 0.018) (0.015 ±0.001) Muscle 0.850 ± 0.112 0.296 ± 0.060 0.115 ± 0.037 0.023 ± 0.003(0.132 ± 0.014) (0.054 ± 0.016) (0.019 ± 0.006) (0.004 ± 0.000) Urine5.258 ± 2.993 26.706 ± 0.900  37.798 ± 2.759  42.009 ± 0.925  Feces0.027 ± 0.002 0.095 ± 0.101 0.097 ± 0.079 1.485 ± 2.389 Carcas 27.525 ±2.159  11.661 ± 0.196  5.563 ± 1.766 1.448 ± 0.256 (0.260 ± 0.042)(0.111 ± 0.008) (0.052 ± 0.017) (0.014 ± 0.002)

TABLE 8 Biodistribution of Tc-99m-peptide 6 in normal rats Upper column:% ID/organ; Lower column: % ID/g (n = 3, mean ± standard deviation)Organs 5 min. 30 min. 60 min. 180 min. Blood 11.205 ± 0.809  3.657 ±1.074 0.936 ± 0.364 0.532 ± 0.421 (1.414 ± 0.176) (0.451 ± 0.052) (0.123± 0.020) (0.062 ± 0.044) Heart 0.275 ± 0.050 0.097 ± 0.026 0.029 ± 0.0080.014 ± 0.006 (0.494 ± 0.083) (0.161 ± 0.029) (0.056 ± 0.017) (0.024 ±0.009) Lungs 1.770 ± 0.274 0.715 ± 0.037 0.369 ± 0.029 0.159 ± 0.023(1.770 ± 0.263) (0.752 ± 0.062) (0.390 ± 0.054) (0.167 ± 0.017) Liver6.374 ± 0.900 4.097 ± 0.758 2.713 ± 0.379 2.167 ± 0.369 (1.003 ± 0.162)(0.607 ± 0.113) (0.465 ± 0.072) (0.353 ± 0.042) Spleen 0.229 ± 0.0500.140 ± 0.040 0.105 ± 0.028 0.090 ± 0.014 (0.548 ± 0.092) (0.198 ±0.083) (0.284 ± 0.054) (0.210 ± 0.011) Kidneys 10.176 ± 2.359  5.916 ±2.411 3.867 ± 1.501 2.634 ± 1.172 (7.546 ± 1.996) (3.971 ± 1.559) (2.975± 1.020) (1.838 ± 0.732) Stomach 0.658 ± 0.196 0.443 ± 0.204 0.166 ±0.037 0.104 ± 0.067 (0.297 ± 0.022) (0.166 ± 0.090) (0.049 ± 0.003)(0.028 ± 0.011) Small 3.680 ± 0.549 3.385 ± 0.898 3.858 ± 0.753 0.996 ±0.574 intestine (0.592 ± 0.073) (0.472 ± 0.117) (0.596 ± 0.120) (0.163 ±0.099) Appendix 0.662 ± 0.009 0.237 ± 0.039 0.097 ± 0.009 2.202 ± 0.468(0.165 ± 0.020) (0.049 ± 0.007) (0.022 ± 0.008) (0.396 ± 0.115) Colon0.311 ± 0.110 0.140 ± 0.087 0.047 ± 0.009 0.031 ± 0.021 (0.625 ± 0.094)(0.223 ± 0.057) (0.094 ± 0.010) (0.069 ± 0.037) Rectum 0.895 ± 0.2450.424 ± 0.151 0.260 ± 0.130 0.051 ± 0.010 (0.913 ± 0.169) (0.503 ±0.372) (0.329 ± 0.245) (0.052 ± 0.009) Adrenal 0.027 ± 0.003 0.011 ±0.001 0.005 ± 0.001 0.002 ± 0.001 (0.668 ± 0.077) (0.276 ± 0.033) (0.117± 0.029) (0.061 ± 0.035) Ovaries 0.096 ± 0.004 0.044 ± 0.003 0.020 ±0.003 0.013 ± 0.009 (1.195 ± 0.048) (0.546 ± 0.040) (0.251 ± 0.044)(0.160 ± 0.117) Bones of 0.578 ± 0.044 0.264 ± 0.014 0.122 ± 0.030 0.067± 0.008 lower limb (0.489 ± 0.065) (0.221 ± 0.014) (0.101 ± 0.023)(0.053 ± 0.005) Skin 1.756 ± 0.161 0.640 ± 0.214 0.238 ± 0.051 0.044 ±0.014 (0.652 ± 0.069) (0.273 ± 0.008) (0.088 ± 0.011) (0.024 ± 0.006)Muscle 1.632 ± 0.201 0.688 ± 0.279 0.180 ± 0.036 0.066 ± 0.009 (0.236 ±0.021) (0.094 ± 0.026) (0.027 ± 0.004) (0.009 ± 0.000) Urine 7.770 ±2.348 56.342 ± 5.430  78.567 ± 2.774  87.408 ± 1.020  Feces 0.048 ±0.016 0.056 ± 0.033 0.025 ± 0.011 0.646 ± 0.636 Carcas 51.858 ± 1.751 22.704 ± 3.734  8.397 ± 1.185 2.772 ± 0.661 (0.465 ± 0.026) (0.199 ±0.024) (0.079 ± 0.014) (0.025 ± 0.007)

TABLE 9 Biodistribution of Tc-99m-peptide 12 in normal rats Uppercolumn: % ID/organ; Lower column: % ID/g (n = 3, mean ± standarddeviation) Organs 5 min. 30 min. 60 min. 180 min. Blood 4.615 ± 0.6110.880 ± 0.059 0.613 ± 0.185 0.332 ± 0.205 (0.683 ± 0.074) (0.146 ±0.019) (0.112 ± 0.038) (0.054 ± 0.035) Heart 0.110 ± 0.016 0.021 ± 0.0040.016 ± 0.006 0.009 ± 0.007 (0.205 ± 0.030) (0.040 ± 0.008) (0.031 ±0.009) (0.018 ± 0.014) Lungs 0.221 ± 0.021 0.065 ± 0.008 0.052 ± 0.0110.030 ± 0.019 (0.259 ± 0.013) (0.073 ± 0.005) (0.061 ± 0.012) (0.036 ±0.025) Liver 32.141 ± 1.082  1.527 ± 0.301 0.696 ± 0.083 0.320 ± 0.221(4.547 ± 0.226) (0.235 ± 0.028) (0.107 ± 0.019) (0.054 ± 0.036) Spleen0.055 ± 0.005 0.019 ± 0.003 0.014 ± 0.002 0.013 ± 0.010 (0.135 ± 0.013)(0.046 ± 0.008) (0.039 ± 0.003) (0.035 ± 0.023) Kidneys 1.380 ± 0.1611.080 ± 0.125 1.043 ± 0.167 1.017 ± 0.639 (1.055 ± 0.152) (0.876 ±0.145) (0.830 ± 0.123) (0.822 ± 0.525) Stomach 0.802 ± 0.643 1.884 ±1.594 1.748 ± 0.191 1.953 ± 1.069 (0.219 ± 0.167) (0.516 ± 0.446) (0.586± 0.076) (0.596 ± 0.178) Small 44.145 ± 2.931  86.860 ± 2.200  86.308 ±1.540  11.574 ± 15.140 intestine (5.915 ± 0.448) (12.285 ± 0.316) (12.392 ± 0.927)  (1.888 ± 2.411) Appendix 0.161 ± 0.22  0.076 ± 0.0030.169 ± 0.047 73.702 ± 24.073 (0.043 ± 0.004) (0.020 ± 0.002) (0.043 ±0.007) (15.226 ± 6.088)  Colon 0.080 ± 0.006 0.023 ± 0.005 0.026 ± 0.0090.048 ± 0.016 (0.167 ± 0.007) (0.048 ± 0.006) (0.046 ± 0.011) (0.112 ±0.052) Rectum 0.162 ± 0.038 0.041 ± 0.011 0.037 ± 0.010 0.019 ± 0.011(0.175 ± 0.003) (0.053 ± 0.005) (0.045 ± 0.011) (0.022 ± 0.016) Adrenal0.009 ± 0.001 0.002 ± 0.000 0.002 ± 0.000 0.001 ± 0.001 (0.223 ± 0.031)(0.057 ± 0.009) (0.054 ± 0.011) (0.235 ± 0.032) Ovaries  0.29 ± 0.0080.008 ± 0.001 0.007 ± 0.001 0.008 ± 0.001 (0.368 ± 0.100) (0.098 ±0.015) (0.088 ± 0.014) (0.038 ± 0.016) Bones of 0.165 ± 0.026 0.057 ±0.005 0.049 ± 0.006 0.029 ± 0.021 lower limb (0.140 ± 0.016) (0.050 ±0.005) (0.043 ± 0.008) (0.025 ± 0.017) Skin 0.374 ± 0.040 0.091 ± 0.0160.104 ± 0.025 0.042 ± 0.047 (0.165 ± 0.027) (0.073 ± 0.008) (0.036 ±0.002) (0.0018 ± 0.019)  Muscle 0.374 ± 0.040 0.137 ± 0.031 0.105 ±0.027 0.047 ± 0.023 (0.165 ± 0.027) (0.021 ± 0.004) (0.016 ± 0.004)(0.007 ± 0.004) Urine 0.400 ± 0.154 2.687 ± 0.285 5.493 ± 0.833 8.767 ±5.591 Feces 0.025 ± 0.002 0.049 ± 0.005 0.105 ± 0.019 0.373 ± 0.122Carcas 14.537 ± 1.466  4.493 ± 0.616 3.411 ± 0.465 1.720 ± 1.276 (0.134± 0.015) (0.041 ± 0.006) (0.031 ± 0.004) (0.016 ± 0.012)

Example 6

Biodistribution in Rat Ulcerative Colitis Model and Usefulness onChronic Inflammation

(1) Method

Preparation of inflammation model: Rat ulcerative colitis model wasprepared according to the method of Anthony et al. (Anthony et al. Int.J. Exp. Path., 76, 215-224, 1995). 2,4,6-Trinitrobenzenesulfonic acid(TNBS) 360 mg was dissolved in ultra pure water 4 ml, and ethanol 3.2 mlwas added therein to prepare 50.0 mg/ml 46% ethanol/physiological salinesolution. A tube was inserted per anum in 7-8 cm depth into theintestine of etherized SD strain rats (Sprague Dawley, specific pathogenfree), female, 7 weeks old, body weight 164-177 g, fasted 24 hoursbefore, and air 0.1 ml was infused. Subsequently, TNBS/46%ethanol/physiological saline 0.2 ml was infused, and massaged and tiltedthe posture for 2 minutes. After 5 days, the rats were used for theexamination.

Tc-99m-peptide 3, Tc-99m-peptide 4, Tc-99m-peptide 6 and prior knownconventional Tc-99m-peptide 12 as a control, about 7.4 MBq/rat each,were administered to the tail vein. After 5 minutes, 30 minutes, 60minutes and 180 minutes from the administration, rats were exsanguinatedand a radioactive distribution in each organ was measured by using a NaIsingle channel analyzer to obtain % ID/each organ and % ID/g organ. Therectum with inflammation region was set as the inflammation region, andratios of [rectum (inflammation)]/[muscle] (ratios of [A]/[M]) andratios of [rectum (inflammation)]/[blood] (ratios of [A]/[B]) wereobtained based on the values of % ID/g organ.

(2) Results

Results are shown in Table 10, Table 11, Table 12 and Table 13.

Tc-99m-peptide 3 showed the ratios of [A]/[M] 3.36±0.58 after 5 minutesfrom the administration, and 7.91±1.16 after 180 minutes from theadministration. High level of radioactivity was exhibited in theinflammation region as compared with the muscle with non-inflammationregion as well as increasing tendency of the ratio of [A]/[M] with thetime course dependent manner. Further, on and after 60 minutes from theadministration, the ratio of [A]/[B] exceeded a value 1, and exhibited2.00±1.50 after 180 minutes from the administration, showing increasingtendency. This may not be due to a non-specific accumulation reflectingincreased blood flow but is considered to be due to a specificaccumulation to inflammation.

Tc-99m-peptide 4 showed the ratios of [A]/[M] 4.37±0.68 after 5 minutesfrom the administration, and 9.29±2.82 after 180 minutes from theadministration. High level of radioactivity was exhibited in theinflammation region as compared with the muscle with non-inflammationregion as well as increasing tendency of the ratio of [A]/[M] with thetime course dependent manner. Further, on and after 60 minutes from theadministration, the ratio of [A]/[B] exceeded a value 1, and exhibited1.51±0.41 after 180 minutes from the administration, showing increasingtendency. This may not be due to a non-specific accumulation reflectingincreased blood flow but is considered to be due to a specificaccumulation to inflammation.

Tc-99m-peptide 6 showed the ratios of [A]/[M] 4.41±0.97 after 5 minutesfrom the administration, and 16.50±11.08 after 180 minutes from theadministration. High level of radioactivity was exhibited in theinflammation region as compared with the muscle with non-inflammationregion as well as increasing tendency of the ratio of [A]/[M] with thetime course dependent manner. Further, on and after 30 minutes from theadministration, the ratio of [A]/[B] exceeded a value 1, and exhibited2.74±1.72 after 180 minutes from the administration, showing increasingtendency. This may not be due to a non-specific accumulation reflectingincreased blood flow but is considered to be due to a specificaccumulation to inflammation.

Tc-99m-peptide 12 showed the ratios of [A]/[M] 4.66±3.13 after 5 minutesfrom the administration, and 6.22±4.61 after 180 minutes from theadministration. This showed a low ratio of [A]/[M] as compared withpeptides of the present invention.

Although the ratio of [A]/[M] showed maximum value, 11.10±12.33, after60 minutes from the administration, it decreased thereafter. Althoughthe ratio of [A]/[B] showed 1.89±2.39 after 60 minutes from theadministration, it decreased thereafter, and was 1.00±0.89 after 180minutes from the administration.

According to the present examination, the peptides of the presentinvention were superior on the points of the accumulation and theretentivity to chronic inflammatory region with high infiltration oflymphocytes and monocytes than the prior known peptide 12, and wereproved to be useful for chronic inflammation such as ulcerative colitis.TABLE 10 Biodistribution of Tc-99m-peptide 3 in rat IBD model Uppercolumn: % ID/organ; Lower column: % ID/g (n = 3, mean ± standarddeviation) Organs 5 min. 30 min. 60 min. 180 min. Blood 6.145 ± 1.4752.397 ± 1.066 0.763 ± 0.238 0.164 ± 0.120 (0.912 ± 0.084) (0.341 ±0.097) (0.108 ± 0.025) (0.023 ± 0.013) Heart 0.0167 ± 0.004  0.045 ±0.006 0.020 ± 0.001 0.004 ± 0.000 (0.345 ± 0.006) (0.101 ± 0.009) (0.042± 0.004) (0.008 ± 0.001) Lungs 0.840 ± 0.027 0.248 ± 0.004 0.151 ± 0.0200.053 ± 0.016 (1.031 ± 0.058) (0.308 ± 0.016) (0.177 ± 0.029) (0.068 ±0.025) Liver 24.382 ± 2.015  16.240 ± 2.633  7.993 ± 1.993 1.249 ± 0.250(4.146 ± 0.528) (2.872 ± 0.793) (1.310 ± 0.313) (0.234 ± 0.053) Spleen0.211 ± 0.008 0.075 ± 0.023 0.074 ± 0.018 0.053 ± 0.016 (0.597 ± 0.046)(0.212 ± 0.070) (0.207 ± 0.037) (0.136 ± 0.043) Kidneys 13.030 ± 1.588 18.869 ± 6.719  16.068 ± 1.238  6.248 ± 0.746 (11.263 ± 1.648)  (15.816± 5.386)  (13.353 ± 1.426)  (5.333 ± 0.482) Stomach 1.428 ± 0.982 0.181± 0.072 0.082 ± 0.013 0.035 ± 0.016 (0.402 ± 0.242) (0.058 ± 0.034)(0.025 ± 0.005) (0.013 ± 0.006) Small 20.876 ± 4.511  34.286 ± 8.601 37.506 ± 2.672  25.538 ± 19.634 intestine (3.056 ± 0.970) (5.520 ±2.262) (5.249 ± 0.446) (4.165 ± 3.176) Appendix 0.405 ± 0.085 0.192 ±0.134 0.080 ± 0.024 14.113 ± 13.789 (0.110 ± 0.031) (0.030 ± 0.003)(0.015 ± 0.004) (3.030 ± 2.871) Colon 0.228 ± 0.044 0.052 ± 0.033 0.046± 0.026 0.087 ± 0.110 (0.484 ± 0.028) (0.140 ± 0.012) (0.083 ± 0.044)(0.229 ± 0.315) Rectum 0.590 ± 0.118 0.286 ± 0.210 0.110 ± 0.062 0.089 ±0.024 (inflammation) (0.573 ± 0.047) (0.203 ± 0.036) (0.097 ± 0.035)(0.041 ± 0.021) Adrenal 0.026 ± 0.003 0.008 ± 0.001 0.003 ± 0.001 0.001± 0.001 (0.294 ± 0.360) (0.097 ± 0.065) (0.067 ± 0.019) (0.082 ± 0.084)Ovaries 0.062 ± 0.046 0.011 ± 0.000 0.005 ± 0.002 0.005 ± 0.005 (0.779 ±0.572) (0.135 ± 0.003) (0.069 ± 0.020) (0.069 ± 0.057) Bones of 0.225 ±0.047 0.087 ± 0.019 0.049 ± 0.018 0.035 ± 0.022 lower limb (0.211 ±0.047) (0.086 ± 0.032) (0.050 ± 0.023) (0.036 ± 0.023) Skin 0.378 ±0.114 0.191 ± 0.211 0.103 ± 0.018 0.069 ± 0.047 (0.220 ± 0.017) (0.102 ±0.049) (0.062 ± 0.041) (0.040 ± 0.034) Muscle 0.537 ± 0.180 0.181 ±0.101 0.094 ± 0.014 0.068 ± 0.049 (0.104 ± 0.008) (0.032 ± 0.015) (0.018± 0.009) (0.014 ± 0.012) Urine 0.681 ± 0.234 4.094 ± 0.769 6.071 ± 0.7968.305 ± 2.667 Feces 0.656 ± 1.023 0.073 ± 0.031 0.073 ± 0.002 0.430 ±0.391 Carcas 16.159 ± 2.511  5.820 ± 1.595 3.392 ± 1.387 2.661 ± 2.059(0.197 ± 0.056) (0.065 ± 0.027) (0.040 ± 0.024) (0.036 ± 0.035) Rectum0.64 ± 0.04 0.63 ± 0.10 1.09 ± 0.70 2.00 ± 1.50 (inflammation)/ bloodRectum 3.36 ± 0.58 4.00 ± 0.95 4.78 ± 1.57 7.91 ± 1.16 (inflammation)/muscle

TABLE 11 Biodistribution of Tc-99m-peptide 4 in rat IBD model Uppercolumn: % ID/organ; Lower column: % ID/g (n = 3, mean ± standarddeviation) Organs 5 min. 30 min. 60 min. 180 min. Blood 5.232 ± 1.6671.764 ± 0.087 0.821 ± 0.308 0.146 ± 0.029 (0.625 ± 0.114) (0.213 ±0.055) (0.092 ± 0.016) (0.019 ± 0.002) Heart 0.132 ± 0.032 0.041 ± 0.0060.017 ± 0.004 0.004 ± 0.002 (0.229 ± 0.058) (0.080 ± 0.019) (0.031 ±0.008) (0.007 ± 0.002) Lungs 0.571 ± 0.230 0.373 ± 0.074 0.171 ± 0.0400.069 ± 0.025 (0.640 ± 0.236) (0.390 ± 0.086) (0.182 ± 0.035) (0.075 ±0.023) Liver 18.757 ± 3.225  13.932 ± 3.695  7.344 ± 0.694 1.219 ± 0.163(2.617 ± 0.132) (1.922 ± 0.413) (1.072 ± 0.231) (0.200 ± 0.024) Spleen0.175 ± 0.088 0.170 ± 0.055 0.081 ± 0.008 0.069 ± 0.031 (0.362 ± 0.051)(0.416 ± 0.168) (0.176 ± 0.046) (0.171 ± 0.53)  Kidneys 4.564 ± 0.8505.646 ± 0.919 5.439 ± 1.647 6.237 ± 1.026 (3.131 ± 0.507) (4.276 ±0.533) (4.262 ± 1.522) (4.490 ± 0.638) Stomach 1.304 ± 1.206 0.209 ±0.097 0.149 ± 0.024 0.090 ± 0.062 (0.323 ± 0.206) (0.057 ± 0.032) (0.039± 0.011) (0.035 ± 0.019) Small 33.890 ± 4.651  41.709 ± 7.462  45.378 ±2.739  30.633 ± 19.329 intestine (4.095 ± 0.762) (5.413 ± 1.168) (6.383± 0.486) (4.459 ± 2.611) Appendix 0.294 ± 0.074 0.094 ± 0.023 0.078 ±0.061 18.242 ± 20.821 (0.060 ± 0.012) (0.022 ± 0.002) (0.016 ± 0.010)(4.248 ± 4.880) Colon 0.181 ± 0.171 0.048 ± 0.015 0.019 ± 0.014 0.053 ±0.066 (0.289 ± 0.072) (0.114 ± 0.047) (0.046 ± 0.010) (0.112 ± 0.135)Rectum 0.410 ± 0.157 0.410 ± 0.398 0.133 ± 0.092 0.032 ± 0.011(inflammation) (0.468 ± 0.127) (0.216 ± 0.150) (0.115 ± 0.076) (0.028 ±0.008) Adrenal 0.015 ± 0.005 0.005 ± 0.002 0.004 ± 0.000 0.001 ± 0.000(0.376 ± 0.119) (0.129 ± 0.061) (0.095 ± 0.008) (0.023 ± 0.008) Ovaries0.042 ± 0.012 0.025 ± 0.011 0.006 ± 0.002 0.002 ± 0.000 (0.529 ± 0.150)(0.309 ± 0.133) (0.080 ± 0.020) (0.022 ± 0.003) Bones of 0.377 ± 0.0540.218 ± 0.052 0.121 ± 0.008 0.078 ± 0.023 lower limb (0.289 ± 0.033)(0.174 ± 0.053) (0.097 ± 0.009) (0.061 ± 0.017) Skin 0.773 ± 0.276 0.327± 0.233 0.112 ± 0.048 0.025 ± 0.011 (0.268 ± 0.050) (0.131 ± 0.047)(0.044 ± 0.007) (0.009 ± 0.002) Muscle 0.757 ± 0.118 0.310 ± 0.157 0.101± 0.012 0.022 ± 0.008 (0.106 ± 0.014) (0.047 ± 0.026) (0.013 ± 0.001) (0.03 ± 0.001) Urine 9.976 ± 3.195 23.885 ± 2.560  35.648 ± 1.482 41.509 ± 2.049  Feces 0.104 ± 0.139 0.032 ± 0.034 0.024 ± 0.023 0.215 ±0.305 Carcas 22.375 ± 2.165  10.901 ± 4.459  4.354 ± 0.670 1.353 ± 0.323(0.192 ± 0.029) (0.101 ± 0.058) (0.037 ± 0.005) (0.011 ± 0.002) Rectum0.74 ± 0.10 0.95 ± 0.42 1.26 ± 0.82 1.51 ± 0.41 (inflammation)/ bloodRectum 4.37 ± 0.68 4.46 ± 1.00 8.81 ± 6.35 9.29 ± 2.82 (inflammation)/muscle

TABLE 12 Biodistribution of Tc-99m-peptide 6 in rat IBD model Uppercolumn: % ID/organ; Lower column: % ID/g (n = 3, mean ± standarddeviation) Organs 5 min. 30 min. 60 min. 180 min. Blood 5.232 ± 1.6671.770 ± 0.092 0.818 ± 0.304 0.146 ± 0.029 (0.625 ± 0.114) (0.214 ±0.057) (0.092 ± 0.016) (0.019 ± 0.002) Heart 0.132 ± 0.032  0.42 ± 0.0060.017 ± 0.004 0.004 ± 0.002 (0.229 ± 0.058) (0.080 ± 0.020) (0.031 ±0.008) (0.007 ± 0.002) Lungs 0.571 ± 0.230 0.374 ± 0.076 0.170 ± 0.0400.069 ± 0.025 (0.640 ± 0.236) (0.391 ± 0.088) (0.181 ± 0.035) (0.075 ±0.023) Liver 18.757 ± 3.225  13.991 ± 3.796  7.322 ± 0.720 1.219 ± 0.163(2.617 ± 0.132) (1.930 ± 0.126) (1.069 ± 0.235) (0.200 ± 0.024) Spleen0.175 ± 0.088 0.171 ± 0.055 0.081 ± 0.008 0.069 ± 0.031 (0.362 ± 0.051)(0.418 ± 0.170) (0.176 ± 0.046) (0.171 ± 0.053) Kidneys 4.564 ± 0.8505.664 ± 0.917 5.427 ± 1.667 6.237 ± 1.026 (3.131 ± 0.507) (4.291 ±0.548) (4.254 ± 1.536) (4.490 ± 0.638) Stomach 1.304 ± 1.206 0.210 ±0.098 0.519 ± 0.649 0.090 ± 0.062 (0.323 ± 0.206) (0.057 ± 0.033) (0.107± 0.109) (0.035 ± 0.019) Small 33.890 ± 4.651  41.389 ± 7.985  45.175 ±2.447  30.633 ± 19.329 intestine (4.095 ± 0.762) (5.371 ± 1.225) (6.357± 0.498) (4.459 ± 2.611) Appendix 0.294 ± 0.074 0.198 ± 0.166 0.078 ±0.061 18.242 ± 20.821 (0.060 ± 0.012) (0.049 ± 0.044) (0.016 ± 0.010)(4.248 ± 4.880) Colon 0.181 ± 0.171 0.048 ± 0.014 0.019 ± 0.014 0.053 ±0.066 (0.289 ± 0.072) (0.115 ± 0.048) (0.046 ± 0.010) (0.112 ± 0.135)Rectum 0.410 ± 0.157 0.413 ± 0.402 0.132 ± 0.091 0.032 ± 0.011(inflammation) (0.468 ± 0.127) (0.217 ± 0.152) (0.114 ± 0.075) (0.028 ±0.008) Adrenal 0.015 ± 0.005 0.005 ± 0.002 0.004 ± 0.000 0.001 ± 0.000(0.376 ± 0.119) (0.130 ± 0.062) (0.094 ± 0.008) (0.023 ± 0.008) Ovaries0.042 ± 0.012 0.025 ± 0.011 0.006 ± 0.002 0.002 ± 0.000 (0.529 ± 0.150)(0.311 ± 0.136) (0.080 ± 0.021) (0.022 ± 0.003) Bones of 0.377 ± 0.0540.219 ± 0.054 0.120 ± 0.008 0.078 ± 0.023 lower limb (0.289 ± 0.033)(0.174 ± 0.054) (0.097 ± 0.010) (0.061 ± 0.017) Skin 0.773 ± 0.276 0.328± 0.237 0.112 ± 0.048 0.025 ± 0.011 (0.268 ± 0.050) (0.131 ± 0.048)(0.044 ± 0.007) (0.009 ± 0.002) Muscle 0.757 ± 0.118 0.311 ± 0.160 0.101± 0.012 0.022 ± 0.008 (0.106 ± 0.014) (0.047 ± 0.026) (0.013 ± 0.001) (0.03 ± 0.001) Urine 9.976 ± 3.195 23.955 ± 2.467  35.537 ± 1.650 41.509 ± 2.049  Feces 0.104 ± 0.139 0.032 ± 0.034 0.024 ± 0.023 0.215 ±0.305 Carcas 22.375 ± 2.165  10.953 ± 4.550  4.338 ± 0.643 1.353 ± 0.323(0.192 ± 0.029) (0.101 ± 0.058) (0.037 ± 0.005) (0.011 ± 0.002) Rectum0.75 ± 0.13 1.27 ± 1.04 1.88 ± 1.44 2.74 ± 1.72 (inflammation)/ bloodRectum 4.41 ± 0.97 5.54 ± 2.99 13.16 ± 11.07 16.50 ± 11.08(inflammation)/ muscle

TABLE 13 Biodistribution of Tc-99m-peptide 12 in rat IBD model Uppercolumn: % ID/organ; Lower column: % ID/g (n = 3, mean ± standarddeviation) Organs 5 min. 30 min. 60 min. 180 min. Blood 10.140 ± 9.197 1.122 ± 0.291 0.954 ± 0.447 0.308 ± 0.193 (2.016 ± 0.074) (0.179 ±0.036) (0.170 ± 0.068) (0.069 ± 0.055) Heart 0.111 ± 0.045 0.027 ± 0.0010.020 ± 0.011 0.007 ± 0.002 (0.269 ± 0.082) (0.061 ± 0.014) (0.049 ±0.026) (0.018 ± 0.009) Lungs 0.280 ± 0.049 0.086 ± 0.017 0.071 ± 0.0320.038 ± 0.029 (0.382 ± 0.067) (0.107 ± 0.032) (0.096 ± 0.049) (0.054 ±0.050) Liver 21.311 ± 7.503  2.012 ± 0.397 0.972 ± 0.399 0.345 ± 0.235(3.326 ± 0.361) (0.322 ± 0.075) (0.162 ± 0.074) (0.059 ± 0.042) Spleen0.072 ± 0.020 0.024 ± 0.006 0.018 ± 0.006 0.009 ± 0.004 (0.189 ± 0.029)(0.064 ± 0.019) (0.053 ± 0.018) (0.031 ± 0.024) Kidneys 1.440 ± 0.3261.077 ± 0.192 1.486 ± 1.002 0.598 ± 0.056 (1.369 ± 0.302) (0.977 ±0.383) (1.377 ± 0.996) (0.506 ± 0.065) Stomach 2.461 ± 2.801 3.446 ±4.540 1.990 ± 0.778 3.500 ± 4.830 (0.631 ± 0.414) (1.050 ± 1.309) (0.659± 0.263) (1.245 ± 1.129) Small 43.689 ± 4.685  82.028 ± 4.176  80.691 ±9.250  82.578 ± 1.469  intestine (6.289 ± 0.568) (12.654 ± 1.252) (13.503 ± 2.964)  (13.121 ± 2.433)  Appendix 0.447 ± 0.366 0.116 ± 0.0190.203 ± 0.070 2.703 ± 3.959 (0.061 ± 0.008) (0.030 ± 0.008) (0.061 ±0.025) (0.783 ± 1.253) Colon 0.194 ± 0.152 0.021 ± 0.001 0.046 ± 0.0320.026 ± 0.024 (0.423 ± 0.333) (0.061 ± 0.009) (0.103 ± 0.077) (0.057 ±0.054) Rectum 1.149 ± 1.318 0.117 ± 0.060 0.637 ± 0.947 0.203 ± 0.328(inflammation) (0.486 ± 0.319) (0.124 ± 0.044) (0.398 ± 0.557) (0.098 ±0.142) Adrenal 0.018 ± 0.002 0.004 ± 0.002 0.017 ± 0.024 0.002 ± 0.003(0.441 ± 0.044) (0.091 ± 0.047) (0.420 ± 0.610) (0.059 ± 0.072) Ovaries0.052 ± 0.027 0.011 ± 0.000 0.015 ± 0.016 0.004 ± 0.004 (0.646 ± 0.334)(0.137 ± 0.004) (0.182 ± 0.197) (0.052 ± 0.050) Bones of 0.225 ± 0.0470.066 ± 0.032 0.066 ± 0.032  0.28 ± 0.020 lower limb (0.211 ± 0.047)(0.078 ± 0.026) (0.062 ± 0.027) (0.028 ± 0.021) Skin 0.378 ± 0.114 0.191± 0.149 0.130 ± 0.048 0.052 ± 0.044 (0.220 ± 0.017) (0.096 ± 0.037)(0.103 ± 0.076) (0.030 ± 0.029) Muscle 0.537 ± 0.180 0.174 ± 0.073 0.120± 0.045 0.052 ± 0.044 (0.104 ± 0.008) (0.030 ± 0.011) (0.026 ± 0.015)(0.011 ± 0.010) Urine 0.681 ± 0.234 3.690 ± 0.886 6.171 ± 0.589 7.135 ±2.769 Feces 0.656 ± 1.023 0.070 ± 0.023 0.356 ± 0.490 0.331 ± 0.325Carcas 16.159 ± 2.511  5.702 ± 1.146 6.036 ± 4.684 2.081 ± 1.769 (0.197± 0.056) (0.062 ± 0.020) (0.070 ± 0.056) (0.027 ± 0.029) Rectum 0.43 ±0.16 0.72 ± 0.33 1.89 ± 2.39 1.00 ± 0.89 (inflammation)/ blood Rectum4.66 ± 3.13 4.21 ± 1.15 11.10 ± 12.33 6.22 ± 4.61 (inflammation)/ muscle

Example 7

Distribution of Tc-99m Labeled Peptide 3 and Peptide 6 in Human Blood

(1) Method

In order to confirm clinical effectiveness of the peptide of the presentinvention in human, binding affinities to leukocytes of two types ofpeptides of the present invention were examined by using human blood.The peptide 3 and the peptide 6 labeled with Tc-99m in Example 2 wereseparated into unlabeled peptides and labeled peptides, and purified byreversed phase HPLC under the same conditions as of HPLC in Example 2. Agradient elution was performed under the following conditions: 20% 80%(0.1% TFA acetonitrile/0.1% TFA water); 0-20 minutes (Radioactivity ofthe peptide 6 after purification was 111 MBq/ml).

The conventionally known peptides, Tc-99m-peptide 11 and Tc-99m-peptide12, were prepared. Each of the peptide 11 and the peptide 12 wasdissolved in dimethylformamide to prepare a solution having theconcentration of 0.1 mg/500 μl. A solution of SnCl₂/10 mM hydrochloricacid (5 mg/10 ml) 50 μl was added to a tartaric acid/PBS solution (5mg/200 μl), and the peptide solution was added thereto. Then ^(99m)-TcO₄⁻ solution (2738 MBq/ml) 0.25 μl was infused immediately thereafter andshaken for several seconds to perform labeling reaction at 120° C. for10 minutes. Final concentration was about 855.6 MBq/125 mg/ml.Purification and measurement of radiochemical purity were performed byusing preparative HPLC. Analysis was performed under the same conditionsas in Example 2, i.e. gradient: 20%→50% (0.1% TFA acetonitrile/0.1% TFAwater); 0→20 minutes. Radioactivity concentration after the purificationwas 287-311 MBq/ml.

Subsequently, a Percoll density gradient solution was prepared based onthe method described in Example 3.

Blood, 20-30 ml, was collected from adult volunteers, 40 years old orless. The Tc-99m labeled peptides, 30 μl (111 MBq/ml, 1.8×10⁻¹¹ mol/mlas Tc-peptide) each, were added thereto and incubated for 30 minutes.The blood sample 2-3 ml was layered over carefully to the preparedPercoll density gradient solution. After centrifugation at 2000 rpm(800×g) for 15 minutes, the tube was frozen and each fraction was cutoff by using a cutter, then the radioactivity of each fraction wasmeasured using Auto-Well Gamma Counter to determine the radioactivitydistribution of Tc-99m-labeled peptides in the blood components.

(2) Results

According to the leukocyte counts of 4100-6100 cells/μl from thehematological parameter of human and the number of receptor FPR,100,000-120,000/cell, found in the reference, estimated numbers of thereceptor FPR in human blood were calculated as 0.68-1.2×10⁻¹² mol/ml,and a ratio of peptide/receptor in human blood was 0.03-0.01. Resultsshowing distributions of 4 types of Tc-99m-peptides and the negativecontrol (Tc-99m-glucoheptonic acid) in the human blood are shown in FIG.18. Results showing percentages of a radioactivity of granulocytefraction to the radioactivity in the total leukocytes, and aradioactivity of lymphocyte and monocyte fraction to the radioactivityin the total leukocytes are shown in Table 14. In Tc-99m-peptide 3,Tc-99m-peptide 11 and Tc-99m-peptide 12, n was 1, and in Tc-99m-peptide6, n was 3.

A radioactivity distribution of Tc-99m-peptide 3 in the granulocytefraction was 21.91% of the radioactivity in the whole blood and that ofTc-99m-peptide 3 in the lymphocyte and monocyte fraction was 39.98% ofthe radioactivity in the whole blood after the incubation for 30minutes. The radioactivity of the granulocyte fraction was 35.41% forthe radioactivity of the whole leukocyte, and the radioactivity of thelymphocyte and monocyte fraction was 64.59% for the radioactivity of thewhole leukocyte. The results demonstrated that in the healthy humanblood, Tc-99m-peptide 3 of the present invention was distributed in thelymphocyte and the monocyte much more than the conventionally knownTc-99m-peptide 11 and Tc-99m-peptide 12.

A radioactivity distribution of Tc-99m-peptide 6 in the granulocytefraction was 29.45% of the radioactivity in the whole blood and that ofTc-99m-peptide 6 in the lymphocyte and monocyte fraction was 6.59% ofthe radioactivity in the whole blood after the incubation for 30minutes. The radioactivity of the granulocyte fraction was 81.94±8.67%for the radioactivity of the whole leukocyte, and the radioactivity ofthe lymphocyte and monocyte fraction was 18.07 ±8.67% for theradioactivity of the whole leukocyte. The results demonstrated that inthe healthy human blood, Tc-99m-peptide 6 of the present invention wasdistributed in the lymphocyte and the monocyte much more than theconventionally known Tc-99m-peptide 11.

A radioactivity distribution of the negative controlTc-99m-glucoheptonic acid in the granulocyte fraction was 1.11% of theradioactivity in the whole blood and that of Tc-99m-glucoheptonic acidin the lymphocyte and monocyte fraction was 2.52% of the radioactivityin the whole blood after the incubation for 30 minutes. In the plasmafraction, 95.39% thereof were distributed. Since it is the negativecontrol which could not bind with leukocytes, a ratio of theradioactivity of the granulocyte fraction and a ratio of theradioactivity of the lymphocyte and monocyte fraction for the totalleukocyte were not calculated.

In the human blood, a radioactivity distribution of Tc-99m-peptide 11 inthe granulocyte fraction was 58.70% of the radioactivity in the wholeblood and that of Tc-99m-peptide 11 in the lymphocyte and monocytefraction was 8.02% of the radioactivity in the whole blood after theincubation for 30 minutes. The radioactivity of the granulocyte fractionwas 87.98% for the radioactivity of the whole leukocyte, and theradioactivity of the lymphocyte and monocyte fraction was 12.03% for theradioactivity of the whole leukocyte.

In the human blood, a radioactivity distribution of Tc-99m-peptide 12 inthe granulocyte fraction was 25.09% of the radioactivity in the wholeblood and that of Tc-99m-peptide 12 in the lymphocyte and monocytefraction was 13.77% of the radioactivity in the whole blood after theincubation for 30 minutes. The radioactivity of the granulocyte fractionwas 64.57% for the radioactivity of the whole leukocyte, and theradioactivity of the lymphocyte and monocyte fraction was 35.43% for theradioactivity of the whole leukocyte.

Above results indicated that the peptide of the present invention wasbound more strongly in the lymphocyte and the monocyte than in thegranulocyte as compared with the negative control Tc-99m-glucoheptonicacid and the conventionally known Tc-99m-peptide 11 or Tc-99m-peptide12. It was also proved that considering the results of Example 4 andExample 6, the peptides of the present invention were effective forchronic inflammation infiltrated with lymphocytes and monocytes. TABLE14 Binding ratio of Tc-99m labeled peptides to human leukocytes (n = 3,mean ± sandard deviation) Binding ratio to leukocytes (%) Lymphocytesand Labeled compound Granulocytes monocytes Tc-99m-peptide 3 35.41 (n= 1) 64.59 (n = 1) Tc-99m-peptide 6 81.94 ± 8.67 18.07 ± 8.67Tc-99m-peptide 11 87.98 (n = 1) 12.03 (n = 1) Tc-99m-peptide 12 64.57 (n= 1) 35.43 (n = 1)

Example 8

Distribution of Tc-99m Labeled Peptide 3 and Peptide 6 in Rat Blood

(1) Method

Preparation of Inflammatory Model:

A model rat of ulcerative colitis was prepared according to the methodof Anthony et al. (Anthony et al. Int. J. Exp. Path., 76, 215-224,1995). 2,4,6-Trinitrobenzene-sulfonic acid (TNBS) 360 mg was dissolvedin ultra pure water 4 ml and ethanol 3.2 ml was added thereto to prepare50.0 mg/ml 46% ethanol/physiological saline. A tube was inserted peranum in 7-8 cm depth into the intestine of etherized SD rat (SpragueDawley strain rat, specific pathogen free), female, 7 weeks age, bodyweight 164-184 g, male, fasted before 24 hours, and air 0.1 ml wasinfused. A solution of TNBS/46% ethanol/physiological saline 0.2 ml wasinfused subsequently and massaged and tilted the posture for 2 minutes.These procedures were repeated for 3 days. After 4 days from the finaladministration, the rat was provided for the experiment, and the bloodwas collected. The collected blood of rat 2 ml was warmed at 37° C. for5 minutes in a warm bath. Each sample 3 μl of Tc-99m-peptide 3 andTc-99m-peptide 6 (111 MBq/ml, Tc-99m-peptide 1.8×10⁻¹¹ mol/ml) purifiedby HPLC was added thereto and incubated for 30 minutes. The blood samplewas carefully layered over onto the previously prepared Percoll densitygradient solution. The layered sample was centrifuged at 2000 rpm(800×g) for 15 minutes. After the centrifugation, the tube was frozenand each fraction was cut off by using a cutter, then the radioactivityof each fraction was measured using Auto-Well Gamma Counter to determinethe radioactivity distributions of Tc-99m-peptide 3 and Tc-99m-peptide 6in each component of the blood.

(2) Results

According to the leukocyte counts 6600-12600 cells/μl from thehematological parameter of female rat and the number of receptor FPR,100,000-120,000/cell, found in the reference, estimated numbers of thereceptor FPR in the blood of rats were calculated as 1.1-2.5×10⁻¹²mol/ml, and ratios of peptide/receptor in rats were 0.02-0.05. Resultsshowing percentages of a radioactivity in each blood component to theradioactivity in the whole blood are shown in FIG. 19. Results showingpercentages of a radioactivity of granulocyte fraction to theradioactivity in the total leukocytes, and a radioactivity of lymphocyteand monocyte fraction for the radioactivity in the total leukocytes areshown in Table 15.

In the blood of rat with ulcerative colitis caused by TNBS, aradioactivity distribution of Tc-99m-peptide 3 in the granulocytefraction was 7.82% of the radioactivity in the whole blood and that ofTc-99m-peptide 3 in the lymphocyte and monocyte fraction was 10.00% ofthe radioactivity in the whole blood after the incubation for 30minutes. The radioactivity of the granulocyte fraction was 43.69% forthe radioactivity of the whole leukocyte, and the radioactivity of thelymphocyte and monocyte fraction was 56.31% for the radioactivity of thewhole leukocyte. A radioactivity distribution of Tc-99m-peptide 6 in thegranulocyte-fraction was 18.34% of the radioactivity in the whole bloodand that of Tc-99m-peptide 6 in the lymphocyte and monocyte fraction was6.57% of the radioactivity in the whole blood after the incubation for30 minutes. The radioactivity of the granulocyte fraction was 74.08% forthe radioactivity of the whole leukocyte, and the radioactivity of thelymphocyte and monocyte fraction was 25.92% for the radioactivity of thewhole leukocyte.

From these results, it has become apparent that Tc-99m-peptide 3 andTc-99m-peptide 6 as a part of the present invention were largelydistributed in the lymphocyte and monocyte fraction in the blood ofulcerative colitis model rats.

From the above, it was proved that the peptide of the present inventionwas effective for diagnosis of chronic inflammation frequentlyinfiltrated lymphocytes and monocytes. TABLE 15 Binding ratio of Tc-99mlabeled peptides to rat leukocytes (Binding ratio to total leukocytes(%) n = 2, mean ± sandard deviation) Binding ratio to leukocytes (%)Lymphocytes and Labeled compound Granulocytes monocytes Tc-99m-peptide 343.69 ± 2.74 56.31 ± 2.74 Tc-99m-peptide 6 74.08 ± 8.37 25.92 ± 8.37

Example 9

Imaging of Tc-99m Labeled Compounds of Peptide 3 and Peptide 6 on RatUlcerative Colitis Model, and Effectiveness on Chronic PhaseInflammation

(1) Method

Preparation of Inflammatory Model:

A model rat of ulcerative colitis was prepared according to the methodof Anthony et al. (Anthony et al. Int. J. Exp. Path., 76, 215-224,1995). 2,4,6-Trinitrobenzene- sulfonic acid (TNBS) 360 mg was dissolvedin ultra pure water 4 ml and ethanol 3.2 ml was added thereto to prepare50.0 mg/ml 46% ethanol/physiological saline. A tube was inserted peranum in 7-8 cm depth into the intestine of etherized SD rat (SpragueDawley strain rat, specific pathogen free), female, 7 weeks age, bodyweight 164-184 g, male, fasted before 24 hours, and air 0.1 ml wasinfused. A solution of TNBS/46% ethanol/physiological saline 0.2 ml wasinfused subsequently, and massaged and tilted the posture for 2 minutes.These procedures were repeated for 3 days. After 4 days from the finaladministration, the rat was provided for the experiment. Tc-99m-peptide3 or Tc-99m-peptide 6 obtained in Example 2, each radioactivity of about37 MBq/rat each, was administered to the tail vein. After 5 minutes, 30minutes, 60 minutes and 120 minutes, the images were recorded by a gammacamera. As a control, Tc-99m labeled leukocytes, which was utilized fordiagnosis of ulcerative colitis in human, was prepared according to themethod of Roca et al. (M. Roca et al. Eur. J. Nucl. Med. 25, 797-799,1998), and administered to the tail vein of the rat in a radioactivityof about 37 MBq/rat, and after 5 minutes, 30 minutes, 60 minutes and 120minutes, the images were recorded by a gamma camera. Tc-99m labeledleukocyte was prepared containing all species of leukocytes such asgranulocytes, lymphocytes and monocytes. After finishing the imaging,i.e. 130 minutes after the administration, the rat was exsanguinatedfrom the abdominal aorta, and each organ was excised. Weights andradioactivities of the excised organs were measured to calculate theradioactivity per g tissue (% ID/g). Further, using the calculatednumerical values, ratios of [inflammation]/[muscle] (ratios of [A]/[M]),ratios of [inflammation]/[blood] (ratios of [A]/[BL]), ratios of[inflammation]/[bone] (ratios of [A]/[BO]), ratios of[inflammation]/[appendix] (ratios of [A]/[AP]), ratios of[inflammation]/[colon] (ratios of [A]/[C]) and ratios of[inflammation]/[rectum] (ratios of [A]/[R]) were determined.

(2) Results

Representative drawings of the obtained results are shown in FIG. 20,FIG. 21 and FIG. 22. Regions of interest are set on the images, andratios of counts in the region of interest of 1 pixel for whole bodycounts (% injection dose (ID)/pixel) are shown in Table 16. Results ofthe ratios indicating [inflammation]/[abdominal background] (ratios of[A]/[BG]) determined from the above ratios are shown in Table 17.Further, % ID/g of the inflammation, ratios of [A]/[M], ratios of[A]/[BL], ratios of [A]/[BO], ratios of [A]/[AP], ratios of [A]/[C] andratios of [A]/[R] obtained from the results of excision are shown inTable 18 and FIG. 23. As a result, in the control Tc-99m labeledleukocytes, % ID/pixel after 30 minutes was 0.11±0.025 (mean±standarddeviation) (n=5), a ratio of [A]/[BG] was 3.21±1.96 (n=5). Further, theradioactivity per g of tissue with inflammation (% ID/g) obtained fromthe exsanguination and the excision after 130 minutes from theadministration was 0.71±0.33, and the ratio of [A]/[BO] was 2.08±1.37and the ratio of [A]/[BL] was 0.25±0.17. The results indicated that thedistribution was larger in the inflammation than in the blood.

Contrary to that, in Tc-99m-peptide 3 of the present invention, %administration/pixel after 30 minutes was 0.043±0.015 (n=5), a ratio of[A]/[BG] was 2.23±0.77 (n=5). Further, the radioactivity per g of tissuewith inflammation (% ID/g) obtained from the exsanguination and theexcision after 130 minutes from the administration was 0.13±0.12, andthe ratio of [A]/[BO] was 3.40±2.78 and the ratio of [A]/[BL] was2.33±2.22. These results indicated that the distribution ofradioactivity was larger in the inflammation than in the blood. Further,in Tc-99m-peptide 6, % ID/pixel after 30 minutes was 0.093±0.048 (n=5),a ratio of [A]/[BG] was 2.15±0.53 (n=5). Further, the radioactivity perg of tissue with inflammation (% ID/g) obtained from the exsanguinationand the excision after 130 minutes from the administration was0.55±0.51, and the ratio of [A]/[BO] was 4.44±2.74 and the ratio of[A]/[BL] was 2.88±1.61. These results indicated that the distribution ofradioactivity was larger in the inflammation than in the bloodindicating the same as in the peptide 3. From the above results, it wasdemonstrated that the Tc-99m labeled peptides of the present inventionwas superior in the depiction of inflammation in organs with largebloodstream such as liver, spleen, heart, kidneys, brain and bone, whichwere easily affected by the blood. In particular, the peptide 6 showedsuperior numerical values in the ratio of [A]/[M], the ratio of[A]/[BL], the ratio of [A]/[BO], the ratio of [A]/[AP], the ratio of[A]/[C] and the ratio of [A]/[R] than the control Tc-99m labeledleukocytes. As a result, it was proved that the peptide of the presentinvention was excellent for depiction in the chronic inflammation,ulcerative colitis. TABLE 16 Accumulation of Tc-99m labeled peptide ininflammation on rat colitis model (% ID/pixel) with or without FMLPinhibition (n = 5, mean ± standard deviation) Elapse of time afteradministration 5 min. 30 min. 1 hr 2 hrs Tc-99m- 0.057 ± 0.043 ± 0.0150.035 ± 0.014 0.031 ± 0.016 peptide 3 0.011 Tc-99m- 0.165 ± 0.093 ±0.048 0.071 ± 0.050 0.069 ± 0.055 peptide 6 0.066 Tc-99m- 0.120 ± 0.111± 0.025 0.102 ± 0.031 0.111 ± 0.043 leukocytes 0.032

TABLE 17 A ratio of inflammation/abdominal background of Tc-99m labeledpeptides and Tc-99m-leukocytes in rat ulcerative colitis model with orwithout FMLP inhibition (n = 5, mean ± standard deviation) Elapse oftime after administration 5 min. 30 min. 1 hr 2 hrs Tc-99m- 1.84 ± 0.412.23 ± 0.77 3.67 ± 1.68 5.87 ± 3.20 peptide 3 Tc-99m- 2.64 ± 0.42 2.15 ±0.53 1.90 ± 0.33 2.73 ± 0.52 peptide 6 Tc-99m- 3.23 ± 1.50 3.21 ± 1.963.23 ± 1.73 3.71 ± 2.17 leukocytes

TABLE 18 Analytical results of Tc-99m labeled peptides andTc-99m-leukocytes on rat ulcerative colitis model by excision (n = 5,mean ± standard deviation) Ratio of Tc-99m- Tc-99m- Tc-99m-inflammation/organ peptide 3 peptide 6 leukocytes Ratio of 10.37 ± 9.54 15.66 ± 11.17 12.12 ± 5.97  inflammation/muscle Ratio of 2.33 ± 2.222.88 ± 1.61 0.25 ± 0.17 inflammation/blood Ratio of 3.40 ± 2.78 4.44 ±2.74 2.08 ± 1.37 inflammation/bone Ratio of 8.75 ± 8.09 13.49 ± 7.75 11.99 ± 4.18  inflammation/appendix Ratio of 2.10 ± 0.83 3.53 ± 2.063.03 ± 2.03 inflammation/colon Ratio of 1.68 ± 0.87 2.79 ± 1.99 2.73 ±1.50 inflammation/rectum Inflammation (% ID/g) 0.13 ± 0.12 0.55 ± 0.510.71 ± 0.33

Example 10

Autoradiography of Tc-99m-Peptide 6 and Tc-99m-Peptide 14 in RatUlcerative Colitis Model and Usefulness Thereof for Chronic Inflammation

(1) Method

A model rat of ulcerative colitis was prepared according to the methodof Anthony et al. (Anthony et al. Int. J. Exp. Path., 76, 215-224,1995). 2,4,6-Trinitrobenzene- sulfonic acid (TNBS) 360 mg was dissolvedin ultra pure water 4 ml and ethanol 3.2 ml was added thereto to prepare50.0 mg/ml 46% ethanol/physiological saline. A tube was inserted peranum in 7-8 cm depth into the intestine of etherized SD rats (SpragueDawley strain rat, specific pathogen free), female, 7 weeks age, bodyweight 164-184 g, male, fasted before 24 hours, and air 0.1 ml wasinfused. A solution of TNBS/46% ethanol/physiological saline 0.2 ml wasinfused subsequently, and massaged and tilted the posture for 2 minutes.These procedures were repeated for 3 days. After 4 days from the finaladministration, the rats were provided for the experiment.Tc-99m-peptide 6 or Tc-99m-peptide 14 obtained in Example 2,radioactivity of about 74 MBq/rat each, was administered to the tailvein, and after 120 minutes, rats were exsanguinated and killed. Afterimmediate excision of the large intestine and removal of the intestinalcontent, areas judged to be inflammation region by macroscopicobservation were embedded in the medium for preparation of frozensection. Immediately thereafter, the embedded samples together withvessels were immersed in the liquid nitrogen for several ten seconds tofreeze the medium containing the excised region of the large intestine.After allowing to stand the sample in the freezer at −20° C. for severalten seconds, frozen sections were prepared by using a cryostat. Afterpreparing sections, the sections were contacted to the imaging plate forautoradiography (Fuji Photo Film Co. Ltd.) for periods from 12 hours to19 hours. Subsequently, the radioactivity distributions were imaged byusing imaging analyzer BAS 2500 (Fuji Photo Film Co. Ltd.). Further, thefrozen sections prepared by the same way were subjected toimmunohistochemical staining for the anti-granulocyte antibody and theanti-monocyte antibody to confirm infiltration of the granulocyte andthe monocyte into the tissue.

(2) Results

Representative drawings of the obtained results are shown in FIG. 24,FIG. 25 and FIG. 26. Regions of interest were set on the images obtainedfrom the results of the autoradiography, and counts per pixel in theregion of interest were calculated and ratios of [inflammation]/[normaltissue] (ratios of [A]/[N]) were determined. Results are shown in FIG.27.

In the rectal region of rats with inflammation, inflammation rangingfrom 2 cm to 4 cm was formed in the whole circular area of theintestinal tract, and the inflammatory regions were observed in allrats. As a result of the immunohistochemical staining, significantinfiltration of granulocytes and monocytes was observed in theinflammation region, and the granulocyte and the monocyte were observedto be distributed in the region corresponding to the inflammation.

Both of Tc-99m-peptide 6 and Tc-99m-peptide 14 showed radioactivitydistributions corresponding to the distributions of the granulocyte andthe monocyte exhibited by the immunostaining in a similar manner asTc-99m-labeled leukocyte. In comparison of the ratios of [A]/[N] in thecontrol Tc-99m-labeled leukocytes, Tc-99m-peptide 6 and Tc-99m-peptide14 obtained from the same section, Tc-99m-peptide 6 and Tc-99m-peptide14 showed higher ratios of [A]/[N] than Tc-99m-labeled leukocyte. As aresult, it was also confirmed that the peptide of the present inventionwas superior in the chronic inflammation such as ulcerative colitis.

Example 11

Assay of Inhibitory Activity of Peptide 3, Peptide 4, Peptide 6, Peptide8, Peptide 9, Peptide 16, Peptide 17 and Peptide 18 for Binding toRecombinant Human Receptor

(1) Method

The experiment was conducted using a recombinant receptor FPR derivedfrom CHO cells (6.24 pmol/ml, 50 mM Tris-HCl, pH 7.4, 10% glycerol, 1%BSA, BioSignal Packard Inc., Amersham Biosciences) and [³H]-FMLP (fMLF,9.25 MBq/2.88-6.25 nmol, Daiichi Pure Chemicals Co. Ltd.). After addingeach peptide to a certain amount of the receptor FPR (0.05 nM, 200pl/well) in concentrations ranging from 10⁻⁴ M to 10⁻¹⁴ M, a certainamount of [³H]-FMLP (0.3 nM, 25 μl/well) was added. After the reaction,an unbound [³H]-FMLP with the receptor FPR and a bound [³H]-FMLP withthe receptor FPR were separated by using GF/C filter. Amount of thebound [³H]-FMLP with the receptor FPR was determined by assaying theradioactivity of [³H]-FMLP bound with the receptor FPR. A concentrationof each peptide inhibiting 50% binding with [³H]-FMLP (IC₅₀) wasdetermined by using the analytical software “Xlfit ver 3.0.3 (CTCLaboratory Systems K.K.)”, further an inhibition constant (Ki) wasdetermined from Kd value of [³H]-FMLP. The tests were repeated threetimes and the assays were repeated three times in each test to determinea mean value.

(2) Results

Results are shown in FIG. 28. Calculated IC₅₀ values and Ki values areshown in Table 19. The control FMLP was calculated to have a Ki value of(2.33+0.45)×10e⁻¹⁰ M. The Ki value of each peptide was calculated asfollows: peptide 3: Ki=(6.50±1.84)×10e⁻⁹ M; peptide 4:Ki=(8.36±3.74)×10e⁻¹⁰ M; peptide 6: Ki=(2.83±1.07)×10e⁻¹⁰ M; peptide 8:Ki=(2.33±0.91)×10e⁻⁹ M; and peptide 9: Ki=(1.28±0.69)×10e⁻¹⁰ M. In thepeptide 16, in which a formyl group was substituted by an acetyl group,the peptide 17, in which a formyl group was substituted by a carbamylgroup, and the peptide 18, in which a formyl group was substituted by amethyl group, the Ki values were (3.74±3.53)×10e⁻⁶ M, (4.24±3.60)×10e⁻⁷M and (3.83±1.12)×10e⁻⁵ M, respectively. As a result, the peptide of thepresent invention was proved to have affinity for the receptor FPR, andbe useful for the diagnosis of inflammation mediated by the leukocytewhich expresses the receptor FPR. TABLE 19 Inhibitory concentration 50(IC₅₀) and inhibition constant (Ki) of each peptide (n = 9, mean ±standard deviation) IC₅₀(M) Ki(M) FMLP (5.67 ± 1.10) × 10e−10 (2.33 ±0.45) × 10e−10 Peptide 3 (1.58 ± 0.45) × 10e−8 (6.50 ± 1.84) × 10e−9Peptide 4 (2.03 ± 0.91) × 10e−9 (8.36 ± 3.74) × 10e−10 Peptide 6 (6.87 ±2.59) × 10e−10 (2.83 ± 1.07) × 10e−10 Peptide 8 (5.65 ± 2.21) × 10e−9(2.33 ± 0.91) × 10e−9 Peptide 9 (3.10 ± 1.69) × 10e−10 (1.28 ± 0.69) ×10e−10 Peptide 12 (7.40 ± 5.03) × 10e−11 (3.05 ± 2.07) × 10e−11 Peptide16 (9.08 ± 8.58) × 10e−6 (3.74 ± 3.53) × 10e−6 Peptide 17 (1.03 ± 0.88)× 10e−6 (4.24 ± 3.60) × 10e−7 Peptide 18 (9.29 ± 2.71) × 10e−5 (3.83 ±1.12) × 10e−5

Example 12

Confirmation of Imaging for Inhibition of Tc-99m-Peptide 6 and Bindingwith Leukocyte In Vivo in Rabbit Infectious Disease Model

(1) Method

Staphylococcus aureus, viable counts about 10⁸, was suspended inphysiological saline 1 ml. The suspension 100 μl was injectedintramuscularly into the right calf of New Zealand White (NZW) strainrabbits, body weight about 2 kg. After elapsing 24 hours, the modelrabbits exhibiting apparent inflammation were anesthetized withpentobarbital. Tc-99m-peptide 6 obtained in Example 2, administrationradioactivity of about 74 MBq, was administered to the auricular vein.After 5 minutes, 1 hour, 2 hours, 3 hours, 4 hours and 5 hours, imageswere recorded by using a gamma camera. A FMLP solution was prepared bydissolving FMLP 1 mg, which was corresponding to about 10,000 times ofthe estimated maximum quantity of the receptor 0.1 nmol/kg, in 5%DMSO/physiological saline. In the FMLP preadministration group, whichwas established for confirming inhibition by FMLP, the FMLP solution wasadministered to the auricular vein 5 minutes before the administrationof Tc-99m-peptide 6. Similar to the group without administering FMLP,images were recorded by using a gamma camera after 5 minutes, 1 hour, 2hours, 3 hours, 4 hours and 5 hours.

(2) Results

Representative figures of the obtained results are shown in FIG. 29 andFIG. 30. Regions of interest are set on the images, and ratios of countsin the region of interest of 1000 pixel for whole body counts (% ID/Kpixel) are shown in Table 20. Ratios indicating [inflammation]/[normalmuscle] (ratios of [A]/[M]) determined from the above ratios are shownin Table 21. As a result, in Tc-99m-peptide 6 without inhibition ofFMLP, accumulation to the inflammation region after 2 hours from theadministration was 1.77±0.25 % ID/Kpixel (mean±standard deviation) (n=3)and increased to 2.62±0.25 %ID/Kpixel after 5 hours from theadministration. The ratio of [A]/[M] also increased from 12.78±6.14after 2 hours to 21.39±5.39 after 5 hours from the administration.Contrary to that, in Tc-99m-peptide 6 with inhibition of FMLP, the ratioof [A]/[M] after 2 hours from the administration was 3.93±0.60, whichwas lower as compared with the case without inhibition of FMLP, and theratio of [A]/[M] after 5 hours from the administration increased to9.05±3.10. However, accumulation to the inflammation region decreasedfrom 0.41±0.10 % ID/Kpixel after 2 hours from the administration to0.30±0.04 % ID/Kpixel after 5 hours from the administration.

These results indicated that the peptide 6 of the present invention wasproved to depict the inflammation region by binding with the receptorFPR existing in the leukocyte. Accumulation of peptide of the presentinvention was thought to indicate onset of inflammation with leukocyteinfiltration. TABLE 20 Accumulation of Tc-99m-peptide 6 in inflammation(% ID/Kpixel) on rabbit infectious disease model with or without FMLPinhibition (n = 3, mean ± standard deviation) Elapse of time afteradministration 5 min. 1 hr 2 hrs 3 hrs 4 hrs 5 hrs Without FMLP 1.74 ±0.22 1.59 ± 0.32 1.77 ± 0.25 2.01 ± 0.29 2.32 ± 0.56 2.62 ± 0.55inhibition With FMLP 2.01 ± 0.43 0.75 ± 0.18 0.41 ± 0.10 0.31 ± 0.060.30 ± 0.03 0.30 ± 0.04 inhibition

TABLE 21 Ratio of inflammation/muscle of Tc-99m-peptide 6 on rabbitinfectious disease model with or without FMLP inhibition (n = 3, mean ±standard deviation) Elapse of time after administration 5 min. 1 hr 2hrs 3 hrs 4 hrs 5 hrs Without 2.25 ± 0.46 6.18 ± 2.99 12.78 ± 6.14 14.53± 5.07 17.87 ± 5.46 21.39 ± 5.39 FMLP inhibition With FMLP 1.81 ± 0.192.62 ± 0.43  3.93 ± 0.60  6.42 ± 1.03  8.58 ± 2.60  9.05 ± 3.10inhibition

INDUSTRIAL APPLICABILITY

According to the present invention, compounds, which exhibit bindingproperties specific to all species of leukocytes, i.e. neutrophils,monocytes and lymphocytes both in vivo and in vitro and can be labeledwith a radioactive metal or a paramagnetic metal, pharmaceuticalcomposition containing the labeled compound as an active ingredientuseful for SPECT image diagnosis, PET image diagnosis and MRI imagediagnosis, can be provided, and the image diagnosis can be performed byimaging a site with vigorous leukocyte infiltration accompanied by animmune reaction in an individual.

1. A compound binding to leukocytes represented by the formula (1):Z-Y-Leu-Phe-(X)_(n)-Lys(NH2)_(m)-ε(—-R-(T)1-U)   (1) (wherein, in theformula (1), Z represents a protecting group for an amino group; Yrepresents Met or Nle; in (X)_(n), X represents a spacer consisting ofone or more of amino acids and/or synthetic organic compounds, and nrepresents 1 or 0; in (NH₂)_(m), NH₂ represents an amide group as aprotecting group for a carboxyl group in the a position of Lys, and mrepresents 1 or 0; in ε(-R-(T)₁-U), R represents Ser or Thr binding toan ε-amino group of Lys through an amide bond, T represents a spacerconsisting of one or more of amino acids and/or synthetic organiccompounds, 1 represents 1 or 0, and U represents a group which can belabeled with a metal; with the proviso that said X and T may be the sameor different from each other).
 2. The compound binding to leukocytesaccording to claim 1, wherein U in the formula (1) is a group consistingof a peptide represented by -Cys-A1-A2 (A1 and A2 are each an amino acidexcept for Cys and Pro), nitrogen-containing cyclic compounds with 8 to20 carbon atoms, nitrogen-containing cyclic carboxylic acid compoundswith 8 to 20 carbon atoms, derivatives of nitrogen-containing cycliccarboxylic acid compounds with 8 to 20 carbon atoms or alkylenaminecarboxylic acids with 4 to 10 carbon atoms, which can be labeled with ametal.
 3. (canceled)
 4. The compound binding to leukocytes according toclaim 1, wherein said compound represented by the formula (1) is oneselected from the group consisting of:formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-Cys-Gly-Asn);formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-Cys-Asp-Asp);formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-Cys-Gly-Asp);formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-Asp-Cys-Asp-Asp);formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraaceticacid); formyl-Nle-Leu-Phe-Lys(NH₂)-ε-(-Ser-D-Ser-Asn-D-Arg-Cys-Asp-Asp);formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-diethylenetriaminepentaacetic acid);formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-1,4,8,11-tetraazacyclotetradecane-butyricacid);formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-Asp-1,4,8,11-tetraazacyclotetradecane-butyricacid);formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Ser-Asn-1,4,8,11-tetraazacyclotetradecane-butyricacid);acetyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-Asp-Cys-Asp-Asp);carbamyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-Asp-Cys-Asp-Asp);and methyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-Asp-Cys-Asp-Asp).5. A medicinal composition containing said compound binding toleukocytes according to claim 1 in labeled state with a radioactivemetal or a paramagnetic metal as the active ingredient.
 6. The medicinalcomposition according to claim 5, wherein said radioactive metal isTc-99m, In-111, Ga-67, CU-64 or Ga-68.
 7. The medicinal compositionaccording to claim 6, wherein said composition is used in SPECT or PETimage diagnosis for imaging a site of vigorous leukocytes infiltrationaccompanied by immune reaction in an individual. 8-9. (canceled)
 10. Themedicinal composition according to claim 5, wherein said paramagneticmetal is Gd, Fe, Mn or Cu.
 11. The medicinal composition according toclaim 10, wherein said composition is used in MRI image diagnosis forimaging a site of vigorous leukocytes infiltration accompanied by immunereaction in an individual.
 12. The medicinal composition according toclaim 5, wherein said radioactive metal is Y-90, Sn- 117m, Sm-1 53,Re-186 or Re-188.
 13. The medicinal composition according to claim 12,wherein said composition is used for the radiotherapy.
 14. The compoundbinding to leukocytes according to claim 2, wherein said compoundrepresented by the formula (1) is one selected from the group consistingof: formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-Cys-Gly-Asn);formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-Cys-Asp-Asp);formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-Cys-Gly-Asp);formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-Asp-Cys-Asp-Asp);formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraaceticacid); formyl-Nle-Leu-Phe-Lys(NH₂)-ε-(-Ser-D-Ser-Asn-D-Arg-Cys-Asp-Asp);formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-diethylenetriaminepentaacetic acid);formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-1,4,8,11-tetraazacyclotetradecane-butyricacid);formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-Asp-1,4,8,11-tetraazacyclotetradecane-butyricacid);formyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Ser-Asn-1,4,8,11-tetraazacyclotetradecane-butyricacid);acetyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-Asp-Cys-Asp-Asp);carbamyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-Asp-Cys-Asp-Asp);and methyl-Nle-Leu-Phe-Nle-Tyr-Lys(NH₂)-ε-(-Ser-D-Arg-Asp-Cys-Asp-Asp).